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What's New from the CLSI Subcommittee on Antimicrobial Susceptibility Testing M100, 29th Edition
Clinical Microbiology N e w s l e t
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203 What’s New from the CLSI Subcommittee on Antimicrobial Susceptibility Testing M100, 29th Edition 210 Primary Toxoplasmosis in Pregnancy: A Case Report and Literature Review
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What’s New from the CLSI Subcommittee on Antimicrobial Susceptibility Testing M100, 29th Edition Taylor C. Abbey, B.S. and Eszter Deak, Ph.D., D(ABMM), Avails Medical, Inc., Menlo Park, California
Abstract The 29th edition of Clinical and Laboratory Standards Institute (CLSI) document M100, Performance Standards for Antimicrobial Susceptibility Testing, was published in January 2019. This edition of the document was corrected in January, February, and May 2019 and revised in March and July 2019. Categorical interpretations were redefined to accommodate the adoption of a susceptible–dosedependent (SDD) category using off-label drug dosages for Enterococcus faecium treated with daptomycin and methicillin-resistant Staphylococcus aureus (MRSA) treated with ceftaroline. Additional revisions included updated breakpoints for disk diffusion and minimum inhibitory concentrations (MICs) for Enterobacteriaceae treated with meropenem-vaborbactam; investigational intended use of cefiderocol breakpoints for Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., and Stenotrophomonas maltophilia; fluoroquinolone disk diffusion and MIC breakpoints for Enterobacteriaceae and P. aeruginosa; and MIC breakpoints for azithromycin to be used with Neisseria gonorrhoeae. A review of these changes and the rationale supporting the changes are presented here, along with the new and updated recommendations, clarifications, and other less significant changes to the M100 document.
Background
Corresponding author: Eszter Deak, Ph.D., D(ABMM), Director of Microbiology, Avails Medical, Inc., Menlo Park, CA. Tel.: 818-324-9072. email: ed@ availsmedical.com
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Antimicrobial susceptibility testing (AST) of clinical isolates is essential for guiding therapy, as well as for surveillance of antimicrobial resistance. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee’s mission is to develop clinical laboratory testing standards that promote accurate AST and appropriate reporting. The Subcommittee is comprised of experts in the field who are continually evaluating breakpoints to determine whether existing minimum inhibitory concentration (MIC) breakpoints continue to be appropriate. These efforts are based on the most current data, including novel resistance mechanisms, a renewed understanding of the pharmacokinetics-pharmacodynamics (PK/PD) of an antimicrobial, or clinical outcome data indicating that the prior breakpoints or the breakpoints under consideration may not be working
adequately. Evaluation of the new data has rendered several drug breakpoints obsolete. The CLSI Performance Standards for Antimicrobial Susceptibility Testing (M100), with all the most current breakpoints for clinically relevant bacteria, are available for free in an online searchable version. Recommendations for selection of the most appropriate agents for testing and reporting of test result interpretation are updated and published regularly to guide practices in the clinical microbiology laboratory and help health care providers prescribe the most effective treatment for their patients. In January each year, CLSI publishes a new edition of its M100 supplement document based on the most recent data. The tables presented in the M100 represent updated information for AST, drug selection, interpretation, and quality control using the procedures in documents M02 (Performance Standards for Antimicrobial Disk Susceptibility
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Tests), M07 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, and M11 (Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria). The MIC or zone diameter values are used to specify breakpoints used to define the clinical categories susceptible (S), intermediate (I), susceptible-dose dependent (SDD), nonsusceptible (NS), and resistant (R). Susceptible implies that the antimicrobial agent used in the recommended dosage to treat the site of infection is clinically effective for that patient’s isolate. Resistant implies that the isolate is not inhibited by the usually achievable drug concentrations, perhaps due to resistance mechanisms, and clinical efficacy has not been shown in treatment studies. Intermediate results indicate clinical efficacy in body sites where the antimicrobial agent is physiologically concentrated (e.g., quinolones and β-lactams in urine). The NS results are reserved for isolates that only have a susceptible interpretive criterion due to the absence or rare occurrence of resistant strains. Under these conditions, a resistant category cannot be established; therefore, a susceptibleonly breakpoint is established. If an organism is placed in the NS category, it does not necessarily mean that the isolate possesses a resistance mechanism; rather, it means that the result does not fall within the interpretive category range of susceptible. Organisms in the NS category should have the identification and AST results confirmed. The recommended MIC and disk diffusion interpretive criteria are based on the usual dosage regimens and routes of administration in the United States. In addition, some of the comments in the tables are marked with an Rx symbol, which indicates that the comment is related to therapy concerns and can be included in a patient report. In 1997, the SDD category was created for the azole class of antifungals used to treat Candida spp., for which multiple FDAapproved dosing regimens were available [1]. An SDD result indicates that the susceptibility of the patient’s isolate depends on the dose and regimen of the antimicrobial agent, and that an altered dosing regimen, usually higher and/or more frequent doses, is needed to reach clinical efficacy. The SDD category is intended to indicate to the physician that maximum allowed doses could be used for treatment when organism MICs fall in this designated range. In January 2014, CLSI M100 adopted the SDD classification for cefepime, a fourth-generation cephalosporin with multiple FDA-approved dosing regimens, for treatment of Enterobacteriaceae [2]. Cefepime provides increased coverage of Gram-negative bacilli in comparison to other cephalosporins approved in the U.S. for clinical therapy due to its chemical side chain substitutes and zwitterionic charge [3]. The rationale for adopting this classification was to provide physicians with an alternative to the broader-spectrum carbapenems for cefepime MICs in the 4- to 8-µg/ml range to further aid in antibiotic stewardship efforts. An SDD category encourages clinicians to use a higher dosing regimen to optimize therapy when an increased MIC result occurs [4]. Given the advantages provided by the SDD category, the CLSI Antimicrobial Susceptibility Testing Committee is interested in evaluating its utility for other drug-bug combinations. Several
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changes were made to the M100 document this year. The two most significant changes were the revision of the daptomycin breakpoints for Enterococcus faecium and the ceftaroline breakpoints for methicillin-resistant Staphylococcus aureus (MRSA), which included SDD categories.
Revised Breakpoints Daptomycin, although not FDA indicated for the treatment of vancomycin-resistant enterococcus (VRE) infections, is one of the few viable drug options for the treatment of E. faecium in serious infections. Ampicillin or vancomycin combined with an aminoglycoside is the preferred treatment for Enterococcus spp. bloodstream infections and endocarditis. Unfortunately, E. faecium is commonly resistant to both vancomycin and high-level aminoglycosides, making it challenging to treat these organisms. The FDAapproved dose of 6 mg/kg of body weight/day is derived from S. aureus infection treatment but is inadequate for the treatment of VRE bloodstream infections. This decision occurred mainly due to the higher MIC values for enterococci (4 µg/ml) versus staphylococci (1 µg/ml), which decreases the probability of achieving optimal PK/PD targets associated with clinical efficacy. Until 2019, CLSI assigned a susceptible MIC daptomycin breakpoint of ≤4 µg/ml to all Enterococcus spp. [5] Enterococcus spp. isolates with MIC values of >4 µg/ml were labeled NS due to the rare occurrence of resistant isolates and the lack of clinical outcome data, precluding a resistant breakpoint. Since the breakpoint was established, reports of daptomycin nonsusceptibility among enterococci have grown, both from patients previously treated with daptomycin [6, 7] and from patients without any prior exposure to daptomycin [8]. Additionally, the occurrence of daptomycin therapy failures has become more prevalent. In January 2019, CLSI created a resistant category of ≥8 µg/ml in response to the new reports. An SDD category was also established based on feasible target attainment at dosing regimens of 6 mg/kg/day and a higher dosing regimen of 12 mg/kg/ day. A wide range of daptomycin doses have been evaluated for the treatment of vancomycin-resistant Enterococcus spp. bloodstream infections [9,10]. According to clinical outcome data, higher offlabel doses of ≥10 mg/kg are associated with lower mortality and better outcomes than the lower dose of 6 mg/kg indicated by the FDA for treatment of S. aureus bloodstream infections [9,10]. However, the January 2019 breakpoints bisected the wild-type E. faecium population, which meant that wild-type isolates generated MICs that were distributed in both the S and SDD categories, resulting in poor categorical agreement. Daptomycin MICs are generally higher for E. faecium than for other enterococcal species. Because daptomycin is predominantly used for E. faecium and not the other enterococcal species, the breakpoints were reassessed following publication and further revised. The Subcommittee on Antimicrobial Susceptibility Testing decided to separate E. faecium from other enterococcal species. For E. faecium, the susceptible category was absorbed by the SDD category due to the inability to reliably distinguish between MICs of 1, 2, and 4 µg/ml [11], and the SDD category was defined as ≤4 µg/ml. Insufficient PK/PD and clinical data in humans prompted a conservative susceptible breakpoint of ≤2 µg/ml for non-E. faecium enterococcal species
and elimination of the SDD category. An intermediate category was introduced to mitigate method or technical variability in susceptibility testing. These updates were published in a revision to CLSI document M100, 29th edition, Table 2D in March 2019 and will be published in the upcoming 30th edition (Table 1). Importantly, this is the first time CLSI has used a non-FDA-approved, off-label dose for SDD. Historically, CLSI categorical definitions have stayed within FDA-approved drug dosing. However, recent recommendations by CLSI, although deemed to be safe upon review of safety data, have gone beyond the dosing currently recommended by the FDA. The rationale is to implement best-practice breakpoints to address urgent clinical needs, including VRE endocarditis and MRSA with elevated ceftaroline MICs. Further work is still needed to determine an accurate and precise testing method for daptomycin and the enterococci given the findings in a recent publication that highlighted the lack of reproducibility in MICs; >20% of the MICs for isolates with high MICs (≥8 µg/ ml), including those recovered from patients with daptomycin treatment failures reported as “S” (MIC ≤ 1 µg/ml) by BMD or gradient diffusion (Etest or MIC test strip) [11]. One particularly troublesome isolate produced MIC results spanning 0.25 µg/ml to 32 µg/ml. Treatment failure was more closely correlated with the presence of mutations in the liaFSR system, a primary resistance mechanism for enterococci to daptomycin [12,13].
with 600 mg q12h. The new SDD breakpoint (2 to 4 µg/ml) is based on a higher dosage of 600 mg q8h over 2 h that is being used outside the United States because places like Latin America and the Asia-Pacific region report higher ceftaroline MICs. The CLSI Subcommittee on Antimicrobial Susceptibility Testing revised the definition in the Breakpoint and Interpretive Category Definitions section in CLSI document M100, 29th edition (page 4) to reflect the broadened criteria for establishing SDD breakpoints. The previous definition of SDD stated that “consideration should be given to the maximum approved dosage regimen” and referred to the drug label. The revised definition indicates that “consideration should be given to the maximum and literature-supported dosage regimens,” indicating that the SDD result may be assigned when “doses well above those used to calculate the susceptible breakpoint are supported by the literature, widely used clinically, and/or approved.” The SDD category enables clinicians to confidently select more narrow-spectrum agents, albeit at a higher dose, to hopefully avoid the development of drug resistance caused by overuse of broad-spectrum antibiotics. To further provide a potential benefit to clinical decision making, the definition of the intermediate (I) category was also revised to indicate that it “implies clinical efficacy in body sites where the drugs are physiologically concentrated,” such as nitrofurantoin in the urine. Table 2 in the upcoming M100 30th edition will be revised to indicate those agents that have the potential to concentrate at an anatomical site with a caret (^). According to the definition, “the intermediate category also includes a buffer zone for inherent variability in test methods, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.” Thus, an unmodified “I” would indicate that the clinician should proceed with caution and likely avoid using the drug [14].
Ceftaroline is used in the treatment of some MRSA infections; however, the FDA changed its approved indications for acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia, and the drug is no longer indicated for treating MRSA infection from those sources. The Subcommittee on Antimicrobial Susceptibility Testing reviewed the ceftaroline breakpoints for S. aureus and made a second SDD recommendation in the 29th M100 edition, again for an off-label dosing regimen. The susceptible breakpoint of ≤1 µg/ml is based on treatment
Table 1. Current daptomycin breakpoints Breakpoint Organism group
S
SDD
E. faecium
b
R
≤4
≥8
≤2b
Enterococcus spp. (not E. faecium) a
I
a
4
≥8
Based on a dosage regimen of 8 to 12 mg/kg q24h. Based on a dosage regimen of 6 mg/kg q24h.
Table 2. Ciprofloxacin and levofloxacin breakpoints Breakpoint Current Organism group
Enterobacteriaceae P. aeruginosa
Historical
Drug
S
I
R
S
I
R
Ciprofloxacin
≤0.25
0.5
≥1
≤1
2
≥4
Levofloxacin
≤0.5
1
≥2
≤2
4
≥8
Ciprofloxacin
≤0.5
1
≥2
≤1
2
≥4
Levofloxacin
≤1
2
≥4
≤2
4
≥8
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CLSI also published revised disk diffusion and MIC breakpoints for the fluoroquinolone class of antibiotics, ciprofloxacin and levofloxacin, for the Enterobacteriaceae other than Salmonella spp. and for Pseudomonas aeruginosa (Table 2) in the M100, 29th edition. Fluoroquinolones are broad-spectrum drugs that come in intravenous (i.v.) and per os (p.o.) formulations, making them an attractive therapeutic option; levofloxacin can even be converted between i.v. and p.o. in a 1:1 ratio. However, resistance to these drugs is emerging. The early breakpoints presented in CLSI Table X (i.e., 10) were discordant with achievable systemic PK that is governed by absorption, distribution, metabolism, and elimination. As a result, an MIC value previously interpreted as susceptible would not meet the PK/PD index of efficacy, even at the highest recommended dosing in healthy patients, much less the most critically ill patients [15]. Tables 2A and 2B-1 were updated to reflect the low probability of treatment success in critically ill patients for a ciprofloxacin MIC of >0.25 µg/ml or >0.5 µg/ml and a levofloxacin MIC of >0.5 µg/ml or >1 µg/ml in Enterobacteriaceae or P. aeruginosa, respectively. As with most updated CLSI breakpoints, the new breakpoints are currently not FDA approved for use on commercial systems. Laboratories can validate the updated breakpoints for off-label use. Another option is to perform reflex testing using disk diffusion or gradient diffusion (e.g., Etest) on isolates with ciprofloxacin MICs of ≤1 µg/ml or levofloxacin MICs of ≤2 µg/ml. Because many isolates will fall under this reflex testing category and implementing reflex testing would become labor-intensive, laboratories may want to think about reserving reflex testing for specific patient populations or specimens obtained from sterile sites. Alternatively, laboratories can perform reflex testing on request only. Another option for laboratories is to attach a report comment to the MIC result.
New Breakpoints In the M100 29th edition, CLSI establishes new breakpoints for the following: (i) disk diffusion and MIC for meropenem-vaborbactam for Enterobacteriaceae; (ii) investigational breakpoints for cefiderocol for Enterobacteriaceae, P. aeruginosa, Acinetobacter spp., and Stenotrophomonas maltophilia; and (iii) MIC breakpoints for azithromycin for N. gonorrhoeae. Meropenem-vaborbactam is one of the newer β-lactam combination drugs and has received FDA clearance for complicated urinary tract infections (UTIs). It primarily inhibits Klebsiella pneumoniae carbapenemases (KPCs), so most clinician requests for the drug will be for carbapenem-resistant Enterobacteriaceae producing a vast majority of molecular class A β-lactamases. Although active against most serine carbapenemases, meropenem-vaborbactam is not active against New Delhi metallo-beta-lactamases and other metallo-beta-lactamase (MBL) carbapenemase producers or OXA carbapenemases and non-Enterobacteriaceae. Meropenem-vaborbactam is already available on some automated susceptibilitytesting platforms, and disks are available for testing. Verification panels are available through Laboratory Specialists and will soon be available through the CDC & FDA Antibiotic Resistance (AR)
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Isolate Bank (https://www.cdc.gov/drugresistance/resistance-bank/ index.html). Meropenem-vaborbactam has been added in Table 1A, Suggested Agents to Test/Report, as one of the β-lactam combination drug options for the Enterobacteriaceae. The β-lactam combination drugs were previously grouped together based on the assumption that they all exhibit similar activities, but they are now listed separately in M100 29th edition based on recognition of their unique activities . Cefiderocol is part of a new class of cephalosporins, called the siderophore cephalosporins, which chelate iron. The drug enters the bacterial cell through active iron transport systems and binds penicillin-binding proteins, thereby disrupting cell wall synthesis. Cefiderocol is active against a wide range of multi-drug-resistant (MDR) Gram-negative bacilli and is recommended for the treatment of complicated UTIs, although the drug is not yet FDA approved for use in the U.S. Until the drug is FDA approved for use in the U.S., CLSI has assigned investigational breakpoints for Enterobacteriaceae, P. aeruginosa, Acinetobacter spp., and S. maltophilia. For broth microdilution methods, cefiderocol susceptibility testing requires the use of iron-depleted media. There are currently no commercial test methods available. A method that requires iron-depleted cation-adjusted Mueller-Hinton broth to determine cefiderocol susceptibilities was approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing in January 2016 [16,17]. This method will be added to M100 as an appendix until it can be incorporated into the M07 document, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Azithromycin MIC breakpoints were established and published for N. gonorrhoeae in the M100, 29th edition. CDC recommends dual therapy with ceftriaxone (250 mg intramuscularly) and azithromycin (1 g p.o.) for the treatment of uncomplicated gonococcal infections of the cervix, urethra, and rectum to cover rare instances of resistance and provide synergistic activity. Most microbiology laboratories perform molecular testing for N. gonorrhoeae, and thus, culture and susceptibility testing are not routinely performed. However, in cases of treatment failure, culture, and susceptibility testing of group A antimicrobials, including azithromycin, ceftriaxone, cefixime, tetracycline, and ciprofloxacin, is recommended. CLSI originally published an Epidemiologic Cutoff Value (ECV) of 1 µg/ml for N. gonorrhoeae and azithromycin in 2017 based on the azithromycin distribution of 15,496 isolates obtained from 2014 to 2016 as part of the CDC Gonococcal Isolate Surveillance Project. In 2018, strains of N. gonorrhoeae with high-level resistance to azithromycin and resistance to ceftriaxone were isolated in the United Kingdom [18] and Australia [19]. A breakpoint was needed to obtain FDA clearance for commercial susceptibility testing for N. gonorrhoeae and azithromycin and to aid in standardized surveillance. The Subcommittee on Antimicrobial Susceptibility Testing set a susceptible-only breakpoint of ≤1 µg/ml based on the wild-type susceptibility distribution. The breakpoint presumes combination therapy. The susceptible breakpoint is supported by clinical data from the 1990s, when azithromycin was used in monotherapy for N. gonorrhoeae infections. Only one
treatment failure was documented, with an azithromycin MIC of 1 µg/ml. Some strains of N. gonorrhoeae with high-level resistance of >256 µg/ml have been associated with treatment failures [18,19]. However, there are insufficient clinical efficacy data for infections caused by isolates with MICs of 2, 4, or 8 µg/ml. In addition to defining a breakpoint for N. gonorrhoeae, disk diffusion and MIC breakpoints for obsolete treatment options, including cefuroxime, cefmetazole, ceftazidime, cefetamet, enoxacin, fleroxacin, lomefloxacin, and ofloxacin, were deleted from Table 2F in the 29th edition of M100. The revised breakpoints for ciprofloxacin and levofloxacin for the Enterobacteriaceae and P. aeruginosa, ceftaroline for S. aureus, and daptomycin for Enterococcus spp. and the new breakpoints for cefiderocol for the Enterobacteriaceae, P. aeruginosa, Acinetobacter spp., and S. maltophilia; meropenem-vaborbactam for the Enterobacteriaceae; and azithromycin for N. gonorrhoeae have been reflected by CLSI in the M100 29th edition table “Breakpoint Additions/Revisions Since 2010.”
Recommendations The M100 29th edition provides a new recommendation advocating the use of colistin as a surrogate for polymyxin B when testing P. aeruginosa, Acinetobacter baumannii, and the Enterobacteriaceae. There are new recommendations for performing MIC testing when specific disk diffusion zone diameters are observed for the Enterobacteriaceae and ceftazidime-avibactam. The current M100 also makes new recommendations for susceptibility testing of Staphylococcus spp. (not S. aureus). CLSI added text to the Surrogate Agents table for colistin (polymyxin E) in the M100 29th edition to indicate that the MICs obtained from testing colistin in broth microdilution (BMD) can be used to predict MICs for polymyxin B for the Enterobacteriaceae, P. aeruginosa, and the A. baumannii complex. Colistin and polymyxin B are members of the polymyxin group of antimicrobial agents. The polymyxins are active against most Gram-negative bacilli. Currently, there are ECVs only for colistin for the Enterobacteriaceae based on MIC distribution data. For P. aeruginosa, there is presently an intermediate breakpoint for polymyxin B. The intermediate breakpoint for colistin for P. aeruginosa was previously deleted and is being reevaluated for polymyxin B. CLSI recommends that colistin be administered with a loading dose at the maximum dose, due to suboptimal PD target attainment rates, and in combination with other antimicrobial agents. In practice, colistin is typically considered salvage therapy and is generally used in combination therapy with other drugs. In some institutions, polymyxin B is used preferentially over colistin. However, testing options for both colistin and polymyxin B are limited to BMD in the absence of surfactant, given that the drugs are made up of large, positively charged molecules that diffuse poorly through agar-based media and adsorb to negatively charged plastics, such as pipette tips and polystyrene tubes and plates. Alternative methods for susceptibility testing of colistin, including the colistin broth disk elution method and the colistin agar screen method [20] have been established and are approved by CLSI.
Resistance to ceftazidime-avibactam may be attributed to the presence of an MBL, mutation of the KPC gene (especially in KPC3), or hyperexpression of KPC, in addition to porin loss. A recent publication found that the disk diffusion method for ceftazidimeavibactam may be overcalling resistance for isolates with zone sizes of 18 to 20 mm [21], producing the appearance of resistance for susceptible isolates. CLSI added a comment to Table 2A for ceftazidime-avibactam to indicate that confirmatory MIC testing should be performed for all isolates that produce disk diffusion zone sizes of 18 to 20 mm. The rationale behind the decision was to avoid unnecessary overuse of broader-spectrum antibiotics. Staphylococcus spp. (not S. aureus) mainly refer to coagulase-negative staphylococci (CoNS). The term CoNS was removed from the Surrogate Agent Tests table for cefoxitin testing and from Tables 1A, 2C, and 3A and replaced with species-specific testing recommendations. In recent years, this group of organisms has become more and more complex with the advent of technology such as matrix-assisted laser desorption ionization–time of flight (MALDITOF), which can identify an array of species. Last year, CLSI created a table listing the most commonly isolated Staphylococcus spp. in respect to the methods used to evaluate oxacillin resistance in the introduction to Table 2C. This table was further modified to reflect species-dependent testing recommendations, with the addition of Staphylococcus epidermidis as a separate species in the M100 29th edition, and to better clarify the method options for identification of oxacillin resistance for each species. The note regarding mecC resistance was modified to indicate that MICs results for mecC are typically categorized as susceptible for cefoxitin resistant and oxacillin. In the M100 29th edition, oxacillin disk diffusion was also added as an option for the identification of oxacillin resistance of S. epidermidis isolates, although many laboratories will likely continue to use the cefoxitin disk or oxacillin MICs. Also, in Table 2C, a separate column was added for “Staphylococcus spp. indications” to define the organisms for which the breakpoints are provided, rather than having the organism names in parentheses under the listed drugs, and oxacillin disk diffusion and MIC breakpoints and cefoxitin disk diffusion have been added for S. epidermidis. Previously, the data for Staphylococcus pseudintermedius and Staphylococcus schleiferi, for which cefoxitin disk diffusion was not a viable option due to an unacceptably high error rate, were reviewed. The option to perform oxacillin disk diffusion testing was reintroduced for these species, as well. For laboratories that do not perform MALDI-TOF, once susceptibility testing, and specifically oxacillin resistance testing, is needed, laboratorians should perform appropriate testing to rule out Staphylococcus lugdunensis and perform oxacillin MIC testing on automated systems.
Clarifications A clarification was made to Table 2A, Zone Diameter and MIC Breakpoints for the Enterobacteriaceae, (p. 37) and the introduction to Tables 3B and 3C (p. 108) to indicate that additional testing, e.g., CarbaNP, mCIM, eCIM, or molecular tests, do not need to be performed once current carbapenem breakpoints have been
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implemented for the Enterobacteriaceae and P. aeruginosa unless the information will be used for epidemiological or infection control purposes. If additional testing is performed, it is also not necessary to edit interpretation results for carbapenems to “R” as long as laboratories are using the current carbapenem breakpoints. Some isolates that produce carbapenemases with a more limited spectrum of activity, such as SME or IMI, may test as susceptible to the cephalosporins. Clarifying guidance has been added to Table 2A for fosfomycin. Fosfomycin has been FDA cleared only for Escherichia coli and Enterococcus faecalis but exhibits good activity against infections by MDR Gram-negative organisms. There is great interest in fosfomycin, which can be administered in an oral sachet formulation, but clinical trials are stalled for the i.v. formulation. The previous comment “for testing and reporting of E. coli urinary tract isolates only” has been expanded to read “disk diffusion and MIC breakpoints apply only to E. coli urinary tract isolates and should not be extrapolated to other species of Enterobacteriaceae.” CLSI recommends that colonies in the disk diffusion zone should not be ignored given the absence of data suggesting clinical insignificance of these colonies.
Nomenclature Changes A couple of taxonomy updates were incorporated into the M100 29th edition. In Table 1C on what to test and report, the “Bacteroides fragilis grp.” was redefined as “Gram-negative anaerobes.” For Table 2J, Breakpoints, the “Bacteroides fragilis grp.” was redefined as “Bacteroides spp. and Parabacteroides spp.”
Intrinsic-Resistance Tables A few changes were made to Appendices B. The Citrobacter amalonaticus group was grouped with Citrobacter koseri, and Klebsiella pneumoniae, Klebsiella oxytoca, and Klebsiella variicola were grouped together. “Raoutella spp.” was added to the intrinsic-resistance table and defined as having intrinsic resistance to ampicillin and ticarcillin. Drugs previously indicated as having intrinsic resistance for the table’s A footnote were added for the Burkholderia cepacia complex.
Information that was Relocated “Using Molecular Assays for Resistance Detection” tables were previously posted on the CLSI website only. These tables have now been published in M100 29th edition as Appendix H to provide a practical approach to help laboratorians troubleshoot and resolve results discrepant between genotypic and phenotypic testing for antimicrobial resistance. Appendix H1 is focused on methicillin (oxacillin) results for S. aureus, Appendix H2 suggests strategies for vancomycin results and Enterococcus spp., and Appendix H3 pertains to extended-spectrum β-lactamases and carbapenemases for Enterobacteriaceae.
Quality Control A new quality control organism, A. baumannii NCTC 13304, was added to Table 4-A2 as a quality control (QC) strain for disk diffusion. Ranges were added for several drugs in Tables 4A-2 and 5A-2 for β-lactam combination agents, many of which are not FDA
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cleared, and for several agents that can be used for an “integrity check” of β-lactamase-producing QC strains. QC ranges were also added for N. gonorrhoeae ATCC 49226 and azithromycin in Table 4B and for P. aeruginosa and meropenem in Table 5A-1.
Summary CLSI continues to make substantial contributions to the field of AST. Several potentially impactful changes were made to the CLSI document in 2019 and clinical laboratories should be aware of the changes and incorporate them into their operating procedures. Moving forward, CLSI should aim to assist in the development of strategies for laboratories to implement the “best practice” breakpoints into their workflow. Over the past decade, CLSI has introduced several non-FDA-approved breakpoints, leading to confusion as to how to proceed, particularly in laboratories without microbiology directors. There remains significant uncertainty among laboratories regarding which antimicrobials and organisms are FDA approved. Several laboratories have only recently transitioned to current cephalosporin and carbapenem breakpoints, while others have decided not to adopt them at all. This includes the cefepime SDD breakpoints. Although the implementation of the SDD category provides promising potential benefits to clinical decision making, current CLSI breakpoints are not yet recognized by the FDA and, in the case of daptomycin for Enterococcus spp. and ceftaroline for MRSA, applies an off-label dose that may never be indicated for use by the FDA as a dosing regimen. FDA clearance is likely not possible for commercial automated AST systems, posing a challenge for breakpoint validation and implementation. Despite this, additional future non-FDA-approved breakpoints are probable as data are reassessed. The off-label use of minocycline for Stenotrophomonas infections and of amoxicillin-clavulanate for anaerobic bacteria is already under discussion. CLSI provides some recommendations through its webinars, but inclusion of detailed guidance on AST platform-specific instructions on how to transition from old breakpoints to new breakpoints and validation instructions, including calculations for categorical agreement (new SDD breakpoints will not be in categorical agreement), how to properly report results, and bridging communication between laboratory and hospital information systems and clinicians, are needed in the M100 document. In January, CLSI created a new working group that will hopefully be able to bridge some of the gaps.
References [1] Rex JH, Pfaller MA, Galgiani JN, Bartlett MS, Espinel-Ingroff A, Ghannoum MA, et al. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections. Clin Infect Dis 1997;24:235-47. [2] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 24th informational supplement 2014; CLSI document M100-S24. Clinical and Laboratory Standards Institute, Wayne, PA. [3] Barnes MD, Taracila MA, Rutter JD, Bethel CR, Galdadas I, Hujer AM, et al. Deciphering the evolution of cephalosporin resistance to ceftolozane-tazobactam in Pseudomonas aeruginosa. mBio 2018;9:e02085-18.
[4] Dudley MN, Ambrose PG, Bhavnani SM, Craig WA, Ferraro MJ, Jones RN, for the Antimicrobial Susceptibility Testing Subcommittee of the Clinical and Laboratory Standards Institute. Background and rationale for revised Clinical and Laboratory Standards Institute interpretive criteria (breakpoints) for Enterobacteriaceae and Pseudomonas aeruginosa. I. Cephalosporins and aztreonam. Clin Infect Dis 2013;56:1301-9. [5] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 28th informational supplement 2018; CLSI document M100-S28. Clinical and Laboratory Standards Institute, Wayne, PA. [6] Munoz-Price LS, Lolans K, Quinn JP. Emergence of resistance to daptomycin during treatment of vancomycin-resistant Enterococcus faecalis infection. Clin Infect Dis 2015;41:565-6. [7] Kelesidis T, Tewhey R, Humphries RM. Evolution of high-level daptomycin resistance in Enterococcus faecium during daptomycin therapy is associated with limited mutations in the bacterial genome. J Antimicrob Chemother 2013;68:1926-8. [8] Kelesidis T, Humphries R, Uslan DZ, Pegues D. De novo daptomycin-nonsusceptible enterococcal infections. Emerg Infect Dis 2012;18:674-6. [9] Chuang YC, Lin HY, Chen PY, Lin CY, Wang JT, Chen YC, et al. Effect of daptomycin dose on the outcome of vancomycin-resistant, daptomycin-susceptible Enterococcus faecium bacteremia. Clin Infect Dis 2017;64:1026-34. [10] Britt NS, Potter EM, Patel N, Steed ME. Comparative effectiveness and safety of standard-, medium-, and high-dose daptomycin strategies for the treatment of vancomycin-resistant enterococcal bacteremia among Veterans Affairs patients. Clin Infect Dis 2017;64:605-13. [11] Campeau SA, Schuetz AN, Kohner P, Arias CA, Hemarajata P, Bard JD, et al. Variability of daptomycin MIC values for Enterococcus faecium when measured by reference broth microdilution and gradient diffusion tests. Antimicrob Agents Chemother 2018;62:e00745-18. [12] Shukla BS, Shelburne S, Reyes K, Kamboj M, Lewis JD, Rincon SL, et al. Influence of minimum inhibitory concentration in clinical outcomes of Enterococcus faecium bacteremia treated with daptomycin: is it time to change the breakpoint? Clin Infect Dis 2016;62:1514-20.
[13] Munita JM, Panesso D, Diaz L, Tran TT, Reyes J, Wanger A, et al. Correlation between mutations in liaFSR of Enterococcus faecium and MIC of daptomycin: revisiting daptomycin breakpoints. Antimicrob Agents Chemother 2012;56:4354-9. [14] Kahlmeter G, Giske CG, Kirn TJ, Sharp SE. Point-counterpoint: difference between the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute recommendations for reporting antimicrobial susceptibility results. J Clin Microbiol 2019;57:e01129-19. [15] Drusano GL, Preston SL, Fowler C, Corrado M, Weisinger B, Kahn J. Relationship between fluoroquinolone area under the curve: minimum inhibitory concentration ratio and the probability of eradication of the infecting pathogen, in patients with nosocomial pneumonia. J Infect Dis 2004;189:1590-7. [16] Clinical and Laboratory Standards Institute. Antimicrobial Susceptibility Testing Meeting Minutes and Presentations, January 2016. [17] Tsuji M, Hackel M, Echols R, Sahm D, Yamano Y. MIC reproducibility of iron-depleted cation adjusted Mueller Hinton broth (IDCAMHB) for microdilution testing of S-649266, a novel siderophore cephalosporin, Abstr Microbe-M-470. In: Abstr 1st Microbe; 2016. [18] Public Health England. UK case of Neisseria gonorrhoeae with highlevel resistance to azithromycin and resistance to ceftriaxone acquired abroad. https://assets.publishing.service.gov.uk/government/uploads/ system/uploads/attachment_data/file/694655/hpr1118_MDRGC. pdf. [Accessed 4 October 2019]. [19] Australian Government. Department of Health. Multi-drug resistant gonorrhea. 2018. http://webarchive.nla.gov.au/gov/20181010021023/ http://www.health.gov.au/internet/main/publishing.nsf/Content/ mr-yr18-dept-dept004.htm. [Accessed 4 October 2019]. [20] Simner PJ, Bergman Y, Trejo M, Roberts AA, Marayan R, Tekle T, et al. Two-site evaluation of the colistin broth disk elution test to determine colistin in vitro activity against gram-negative bacilli. J Clin Microbiol 2019;57:e01163-18. [21] Shields RK, Clancy CJ, Pasculle AW, Press EG, Haidar G, Hao B. Verification of ceftazidime-avibactam and ceftolozane-tazobactam susceptibility testing methods against carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa. J Clin Microbiol 2018;56:e01093-17.
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What’s New from the CLSI Subcommittee on Antimicrobial Susceptibility Testing M100, 29th Edition Taylor C. Abbey, B.S. and Eszter Deak, Ph.D., D(ABMM), Avails Medical, Inc., Menlo Park, California
Abstract The 29th edition of Clinical and Laboratory Standards Institute (CLSI) document M100, Performance Standards for Antimicrobial Susceptibility Testing, was published in January 2019. This edition of the document was corrected in January, February, and May 2019 and revised in March and July 2019. Categorical interpretations were redefined to accommodate the adoption of a susceptible–dosedependent (SDD) category using off-label drug dosages for Enterococcus faecium treated with daptomycin and methicillin-resistant Staphylococcus aureus (MRSA) treated with ceftaroline. Additional revisions included updated breakpoints for disk diffusion and minimum inhibitory concentrations (MICs) for Enterobacteriaceae treated with meropenem-vaborbactam; investigational intended use of cefiderocol breakpoints for Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., and Stenotrophomonas maltophilia; fluoroquinolone disk diffusion and MIC breakpoints for Enterobacteriaceae and P. aeruginosa; and MIC breakpoints for azithromycin to be used with Neisseria gonorrhoeae. A review of these changes and the rationale supporting the changes are presented here, along with the new and updated recommendations, clarifications, and other less significant changes to the M100 document.
Background
Corresponding author: Eszter Deak, Ph.D., D(ABMM), Director of Microbiology, Avails Medical, Inc., Menlo Park, CA. Tel.: 818-324-9072. email: ed@ availsmedical.com
CMN
Antimicrobial susceptibility testing (AST) of clinical isolates is essential for guiding therapy, as well as for surveillance of antimicrobial resistance. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee’s mission is to develop clinical laboratory testing standards that promote accurate AST and appropriate reporting. The Subcommittee is comprised of experts in the field who are continually evaluating breakpoints to determine whether existing minimum inhibitory concentration (MIC) breakpoints continue to be appropriate. These efforts are based on the most current data, including novel resistance mechanisms, a renewed understanding of the pharmacokinetics-pharmacodynamics (PK/PD) of an antimicrobial, or clinical outcome data indicating that the prior breakpoints or the breakpoints under consideration may not be working
adequately. Evaluation of the new data has rendered several drug breakpoints obsolete. The CLSI Performance Standards for Antimicrobial Susceptibility Testing (M100), with all the most current breakpoints for clinically relevant bacteria, are available for free in an online searchable version. Recommendations for selection of the most appropriate agents for testing and reporting of test result interpretation are updated and published regularly to guide practices in the clinical microbiology laboratory and help health care providers prescribe the most effective treatment for their patients. In January each year, CLSI publishes a new edition of its M100 supplement document based on the most recent data. The tables presented in the M100 represent updated information for AST, drug selection, interpretation, and quality control using the procedures in documents M02 (Performance Standards for Antimicrobial Disk Susceptibility
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Tests), M07 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, and M11 (Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria). The MIC or zone diameter values are used to specify breakpoints used to define the clinical categories susceptible (S), intermediate (I), susceptible-dose dependent (SDD), nonsusceptible (NS), and resistant (R). Susceptible implies that the antimicrobial agent used in the recommended dosage to treat the site of infection is clinically effective for that patient’s isolate. Resistant implies that the isolate is not inhibited by the usually achievable drug concentrations, perhaps due to resistance mechanisms, and clinical efficacy has not been shown in treatment studies. Intermediate results indicate clinical efficacy in body sites where the antimicrobial agent is physiologically concentrated (e.g., quinolones and β-lactams in urine). The NS results are reserved for isolates that only have a susceptible interpretive criterion due to the absence or rare occurrence of resistant strains. Under these conditions, a resistant category cannot be established; therefore, a susceptibleonly breakpoint is established. If an organism is placed in the NS category, it does not necessarily mean that the isolate possesses a resistance mechanism; rather, it means that the result does not fall within the interpretive category range of susceptible. Organisms in the NS category should have the identification and AST results confirmed. The recommended MIC and disk diffusion interpretive criteria are based on the usual dosage regimens and routes of administration in the United States. In addition, some of the comments in the tables are marked with an Rx symbol, which indicates that the comment is related to therapy concerns and can be included in a patient report. In 1997, the SDD category was created for the azole class of antifungals used to treat Candida spp., for which multiple FDAapproved dosing regimens were available [1]. An SDD result indicates that the susceptibility of the patient’s isolate depends on the dose and regimen of the antimicrobial agent, and that an altered dosing regimen, usually higher and/or more frequent doses, is needed to reach clinical efficacy. The SDD category is intended to indicate to the physician that maximum allowed doses could be used for treatment when organism MICs fall in this designated range. In January 2014, CLSI M100 adopted the SDD classification for cefepime, a fourth-generation cephalosporin with multiple FDA-approved dosing regimens, for treatment of Enterobacteriaceae [2]. Cefepime provides increased coverage of Gram-negative bacilli in comparison to other cephalosporins approved in the U.S. for clinical therapy due to its chemical side chain substitutes and zwitterionic charge [3]. The rationale for adopting this classification was to provide physicians with an alternative to the broader-spectrum carbapenems for cefepime MICs in the 4- to 8-µg/ml range to further aid in antibiotic stewardship efforts. An SDD category encourages clinicians to use a higher dosing regimen to optimize therapy when an increased MIC result occurs [4]. Given the advantages provided by the SDD category, the CLSI Antimicrobial Susceptibility Testing Committee is interested in evaluating its utility for other drug-bug combinations. Several
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changes were made to the M100 document this year. The two most significant changes were the revision of the daptomycin breakpoints for Enterococcus faecium and the ceftaroline breakpoints for methicillin-resistant Staphylococcus aureus (MRSA), which included SDD categories.
Revised Breakpoints Daptomycin, although not FDA indicated for the treatment of vancomycin-resistant enterococcus (VRE) infections, is one of the few viable drug options for the treatment of E. faecium in serious infections. Ampicillin or vancomycin combined with an aminoglycoside is the preferred treatment for Enterococcus spp. bloodstream infections and endocarditis. Unfortunately, E. faecium is commonly resistant to both vancomycin and high-level aminoglycosides, making it challenging to treat these organisms. The FDAapproved dose of 6 mg/kg of body weight/day is derived from S. aureus infection treatment but is inadequate for the treatment of VRE bloodstream infections. This decision occurred mainly due to the higher MIC values for enterococci (4 µg/ml) versus staphylococci (1 µg/ml), which decreases the probability of achieving optimal PK/PD targets associated with clinical efficacy. Until 2019, CLSI assigned a susceptible MIC daptomycin breakpoint of ≤4 µg/ml to all Enterococcus spp. [5] Enterococcus spp. isolates with MIC values of >4 µg/ml were labeled NS due to the rare occurrence of resistant isolates and the lack of clinical outcome data, precluding a resistant breakpoint. Since the breakpoint was established, reports of daptomycin nonsusceptibility among enterococci have grown, both from patients previously treated with daptomycin [6, 7] and from patients without any prior exposure to daptomycin [8]. Additionally, the occurrence of daptomycin therapy failures has become more prevalent. In January 2019, CLSI created a resistant category of ≥8 µg/ml in response to the new reports. An SDD category was also established based on feasible target attainment at dosing regimens of 6 mg/kg/day and a higher dosing regimen of 12 mg/kg/ day. A wide range of daptomycin doses have been evaluated for the treatment of vancomycin-resistant Enterococcus spp. bloodstream infections [9,10]. According to clinical outcome data, higher offlabel doses of ≥10 mg/kg are associated with lower mortality and better outcomes than the lower dose of 6 mg/kg indicated by the FDA for treatment of S. aureus bloodstream infections [9,10]. However, the January 2019 breakpoints bisected the wild-type E. faecium population, which meant that wild-type isolates generated MICs that were distributed in both the S and SDD categories, resulting in poor categorical agreement. Daptomycin MICs are generally higher for E. faecium than for other enterococcal species. Because daptomycin is predominantly used for E. faecium and not the other enterococcal species, the breakpoints were reassessed following publication and further revised. The Subcommittee on Antimicrobial Susceptibility Testing decided to separate E. faecium from other enterococcal species. For E. faecium, the susceptible category was absorbed by the SDD category due to the inability to reliably distinguish between MICs of 1, 2, and 4 µg/ml [11], and the SDD category was defined as ≤4 µg/ml. Insufficient PK/PD and clinical data in humans prompted a conservative susceptible breakpoint of ≤2 µg/ml for non-E. faecium enterococcal species
and elimination of the SDD category. An intermediate category was introduced to mitigate method or technical variability in susceptibility testing. These updates were published in a revision to CLSI document M100, 29th edition, Table 2D in March 2019 and will be published in the upcoming 30th edition (Table 1). Importantly, this is the first time CLSI has used a non-FDA-approved, off-label dose for SDD. Historically, CLSI categorical definitions have stayed within FDA-approved drug dosing. However, recent recommendations by CLSI, although deemed to be safe upon review of safety data, have gone beyond the dosing currently recommended by the FDA. The rationale is to implement best-practice breakpoints to address urgent clinical needs, including VRE endocarditis and MRSA with elevated ceftaroline MICs. Further work is still needed to determine an accurate and precise testing method for daptomycin and the enterococci given the findings in a recent publication that highlighted the lack of reproducibility in MICs; >20% of the MICs for isolates with high MICs (≥8 µg/ ml), including those recovered from patients with daptomycin treatment failures reported as “S” (MIC ≤ 1 µg/ml) by BMD or gradient diffusion (Etest or MIC test strip) [11]. One particularly troublesome isolate produced MIC results spanning 0.25 µg/ml to 32 µg/ml. Treatment failure was more closely correlated with the presence of mutations in the liaFSR system, a primary resistance mechanism for enterococci to daptomycin [12,13].
with 600 mg q12h. The new SDD breakpoint (2 to 4 µg/ml) is based on a higher dosage of 600 mg q8h over 2 h that is being used outside the United States because places like Latin America and the Asia-Pacific region report higher ceftaroline MICs. The CLSI Subcommittee on Antimicrobial Susceptibility Testing revised the definition in the Breakpoint and Interpretive Category Definitions section in CLSI document M100, 29th edition (page 4) to reflect the broadened criteria for establishing SDD breakpoints. The previous definition of SDD stated that “consideration should be given to the maximum approved dosage regimen” and referred to the drug label. The revised definition indicates that “consideration should be given to the maximum and literature-supported dosage regimens,” indicating that the SDD result may be assigned when “doses well above those used to calculate the susceptible breakpoint are supported by the literature, widely used clinically, and/or approved.” The SDD category enables clinicians to confidently select more narrow-spectrum agents, albeit at a higher dose, to hopefully avoid the development of drug resistance caused by overuse of broad-spectrum antibiotics. To further provide a potential benefit to clinical decision making, the definition of the intermediate (I) category was also revised to indicate that it “implies clinical efficacy in body sites where the drugs are physiologically concentrated,” such as nitrofurantoin in the urine. Table 2 in the upcoming M100 30th edition will be revised to indicate those agents that have the potential to concentrate at an anatomical site with a caret (^). According to the definition, “the intermediate category also includes a buffer zone for inherent variability in test methods, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.” Thus, an unmodified “I” would indicate that the clinician should proceed with caution and likely avoid using the drug [14].
Ceftaroline is used in the treatment of some MRSA infections; however, the FDA changed its approved indications for acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia, and the drug is no longer indicated for treating MRSA infection from those sources. The Subcommittee on Antimicrobial Susceptibility Testing reviewed the ceftaroline breakpoints for S. aureus and made a second SDD recommendation in the 29th M100 edition, again for an off-label dosing regimen. The susceptible breakpoint of ≤1 µg/ml is based on treatment
Table 1. Current daptomycin breakpoints Breakpoint Organism group
S
SDD
E. faecium
b
R
≤4
≥8
≤2b
Enterococcus spp. (not E. faecium) a
I
a
4
≥8
Based on a dosage regimen of 8 to 12 mg/kg q24h. Based on a dosage regimen of 6 mg/kg q24h.
Table 2. Ciprofloxacin and levofloxacin breakpoints Breakpoint Current Organism group
Enterobacteriaceae P. aeruginosa
Historical
Drug
S
I
R
S
I
R
Ciprofloxacin
≤0.25
0.5
≥1
≤1
2
≥4
Levofloxacin
≤0.5
1
≥2
≤2
4
≥8
Ciprofloxacin
≤0.5
1
≥2
≤1
2
≥4
Levofloxacin
≤1
2
≥4
≤2
4
≥8
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CLSI also published revised disk diffusion and MIC breakpoints for the fluoroquinolone class of antibiotics, ciprofloxacin and levofloxacin, for the Enterobacteriaceae other than Salmonella spp. and for Pseudomonas aeruginosa (Table 2) in the M100, 29th edition. Fluoroquinolones are broad-spectrum drugs that come in intravenous (i.v.) and per os (p.o.) formulations, making them an attractive therapeutic option; levofloxacin can even be converted between i.v. and p.o. in a 1:1 ratio. However, resistance to these drugs is emerging. The early breakpoints presented in CLSI Table X (i.e., 10) were discordant with achievable systemic PK that is governed by absorption, distribution, metabolism, and elimination. As a result, an MIC value previously interpreted as susceptible would not meet the PK/PD index of efficacy, even at the highest recommended dosing in healthy patients, much less the most critically ill patients [15]. Tables 2A and 2B-1 were updated to reflect the low probability of treatment success in critically ill patients for a ciprofloxacin MIC of >0.25 µg/ml or >0.5 µg/ml and a levofloxacin MIC of >0.5 µg/ml or >1 µg/ml in Enterobacteriaceae or P. aeruginosa, respectively. As with most updated CLSI breakpoints, the new breakpoints are currently not FDA approved for use on commercial systems. Laboratories can validate the updated breakpoints for off-label use. Another option is to perform reflex testing using disk diffusion or gradient diffusion (e.g., Etest) on isolates with ciprofloxacin MICs of ≤1 µg/ml or levofloxacin MICs of ≤2 µg/ml. Because many isolates will fall under this reflex testing category and implementing reflex testing would become labor-intensive, laboratories may want to think about reserving reflex testing for specific patient populations or specimens obtained from sterile sites. Alternatively, laboratories can perform reflex testing on request only. Another option for laboratories is to attach a report comment to the MIC result.
New Breakpoints In the M100 29th edition, CLSI establishes new breakpoints for the following: (i) disk diffusion and MIC for meropenem-vaborbactam for Enterobacteriaceae; (ii) investigational breakpoints for cefiderocol for Enterobacteriaceae, P. aeruginosa, Acinetobacter spp., and Stenotrophomonas maltophilia; and (iii) MIC breakpoints for azithromycin for N. gonorrhoeae. Meropenem-vaborbactam is one of the newer β-lactam combination drugs and has received FDA clearance for complicated urinary tract infections (UTIs). It primarily inhibits Klebsiella pneumoniae carbapenemases (KPCs), so most clinician requests for the drug will be for carbapenem-resistant Enterobacteriaceae producing a vast majority of molecular class A β-lactamases. Although active against most serine carbapenemases, meropenem-vaborbactam is not active against New Delhi metallo-beta-lactamases and other metallo-beta-lactamase (MBL) carbapenemase producers or OXA carbapenemases and non-Enterobacteriaceae. Meropenem-vaborbactam is already available on some automated susceptibilitytesting platforms, and disks are available for testing. Verification panels are available through Laboratory Specialists and will soon be available through the CDC & FDA Antibiotic Resistance (AR)
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Isolate Bank (https://www.cdc.gov/drugresistance/resistance-bank/ index.html). Meropenem-vaborbactam has been added in Table 1A, Suggested Agents to Test/Report, as one of the β-lactam combination drug options for the Enterobacteriaceae. The β-lactam combination drugs were previously grouped together based on the assumption that they all exhibit similar activities, but they are now listed separately in M100 29th edition based on recognition of their unique activities . Cefiderocol is part of a new class of cephalosporins, called the siderophore cephalosporins, which chelate iron. The drug enters the bacterial cell through active iron transport systems and binds penicillin-binding proteins, thereby disrupting cell wall synthesis. Cefiderocol is active against a wide range of multi-drug-resistant (MDR) Gram-negative bacilli and is recommended for the treatment of complicated UTIs, although the drug is not yet FDA approved for use in the U.S. Until the drug is FDA approved for use in the U.S., CLSI has assigned investigational breakpoints for Enterobacteriaceae, P. aeruginosa, Acinetobacter spp., and S. maltophilia. For broth microdilution methods, cefiderocol susceptibility testing requires the use of iron-depleted media. There are currently no commercial test methods available. A method that requires iron-depleted cation-adjusted Mueller-Hinton broth to determine cefiderocol susceptibilities was approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing in January 2016 [16,17]. This method will be added to M100 as an appendix until it can be incorporated into the M07 document, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Azithromycin MIC breakpoints were established and published for N. gonorrhoeae in the M100, 29th edition. CDC recommends dual therapy with ceftriaxone (250 mg intramuscularly) and azithromycin (1 g p.o.) for the treatment of uncomplicated gonococcal infections of the cervix, urethra, and rectum to cover rare instances of resistance and provide synergistic activity. Most microbiology laboratories perform molecular testing for N. gonorrhoeae, and thus, culture and susceptibility testing are not routinely performed. However, in cases of treatment failure, culture, and susceptibility testing of group A antimicrobials, including azithromycin, ceftriaxone, cefixime, tetracycline, and ciprofloxacin, is recommended. CLSI originally published an Epidemiologic Cutoff Value (ECV) of 1 µg/ml for N. gonorrhoeae and azithromycin in 2017 based on the azithromycin distribution of 15,496 isolates obtained from 2014 to 2016 as part of the CDC Gonococcal Isolate Surveillance Project. In 2018, strains of N. gonorrhoeae with high-level resistance to azithromycin and resistance to ceftriaxone were isolated in the United Kingdom [18] and Australia [19]. A breakpoint was needed to obtain FDA clearance for commercial susceptibility testing for N. gonorrhoeae and azithromycin and to aid in standardized surveillance. The Subcommittee on Antimicrobial Susceptibility Testing set a susceptible-only breakpoint of ≤1 µg/ml based on the wild-type susceptibility distribution. The breakpoint presumes combination therapy. The susceptible breakpoint is supported by clinical data from the 1990s, when azithromycin was used in monotherapy for N. gonorrhoeae infections. Only one
treatment failure was documented, with an azithromycin MIC of 1 µg/ml. Some strains of N. gonorrhoeae with high-level resistance of >256 µg/ml have been associated with treatment failures [18,19]. However, there are insufficient clinical efficacy data for infections caused by isolates with MICs of 2, 4, or 8 µg/ml. In addition to defining a breakpoint for N. gonorrhoeae, disk diffusion and MIC breakpoints for obsolete treatment options, including cefuroxime, cefmetazole, ceftazidime, cefetamet, enoxacin, fleroxacin, lomefloxacin, and ofloxacin, were deleted from Table 2F in the 29th edition of M100. The revised breakpoints for ciprofloxacin and levofloxacin for the Enterobacteriaceae and P. aeruginosa, ceftaroline for S. aureus, and daptomycin for Enterococcus spp. and the new breakpoints for cefiderocol for the Enterobacteriaceae, P. aeruginosa, Acinetobacter spp., and S. maltophilia; meropenem-vaborbactam for the Enterobacteriaceae; and azithromycin for N. gonorrhoeae have been reflected by CLSI in the M100 29th edition table “Breakpoint Additions/Revisions Since 2010.”
Recommendations The M100 29th edition provides a new recommendation advocating the use of colistin as a surrogate for polymyxin B when testing P. aeruginosa, Acinetobacter baumannii, and the Enterobacteriaceae. There are new recommendations for performing MIC testing when specific disk diffusion zone diameters are observed for the Enterobacteriaceae and ceftazidime-avibactam. The current M100 also makes new recommendations for susceptibility testing of Staphylococcus spp. (not S. aureus). CLSI added text to the Surrogate Agents table for colistin (polymyxin E) in the M100 29th edition to indicate that the MICs obtained from testing colistin in broth microdilution (BMD) can be used to predict MICs for polymyxin B for the Enterobacteriaceae, P. aeruginosa, and the A. baumannii complex. Colistin and polymyxin B are members of the polymyxin group of antimicrobial agents. The polymyxins are active against most Gram-negative bacilli. Currently, there are ECVs only for colistin for the Enterobacteriaceae based on MIC distribution data. For P. aeruginosa, there is presently an intermediate breakpoint for polymyxin B. The intermediate breakpoint for colistin for P. aeruginosa was previously deleted and is being reevaluated for polymyxin B. CLSI recommends that colistin be administered with a loading dose at the maximum dose, due to suboptimal PD target attainment rates, and in combination with other antimicrobial agents. In practice, colistin is typically considered salvage therapy and is generally used in combination therapy with other drugs. In some institutions, polymyxin B is used preferentially over colistin. However, testing options for both colistin and polymyxin B are limited to BMD in the absence of surfactant, given that the drugs are made up of large, positively charged molecules that diffuse poorly through agar-based media and adsorb to negatively charged plastics, such as pipette tips and polystyrene tubes and plates. Alternative methods for susceptibility testing of colistin, including the colistin broth disk elution method and the colistin agar screen method [20] have been established and are approved by CLSI.
Resistance to ceftazidime-avibactam may be attributed to the presence of an MBL, mutation of the KPC gene (especially in KPC3), or hyperexpression of KPC, in addition to porin loss. A recent publication found that the disk diffusion method for ceftazidimeavibactam may be overcalling resistance for isolates with zone sizes of 18 to 20 mm [21], producing the appearance of resistance for susceptible isolates. CLSI added a comment to Table 2A for ceftazidime-avibactam to indicate that confirmatory MIC testing should be performed for all isolates that produce disk diffusion zone sizes of 18 to 20 mm. The rationale behind the decision was to avoid unnecessary overuse of broader-spectrum antibiotics. Staphylococcus spp. (not S. aureus) mainly refer to coagulase-negative staphylococci (CoNS). The term CoNS was removed from the Surrogate Agent Tests table for cefoxitin testing and from Tables 1A, 2C, and 3A and replaced with species-specific testing recommendations. In recent years, this group of organisms has become more and more complex with the advent of technology such as matrix-assisted laser desorption ionization–time of flight (MALDITOF), which can identify an array of species. Last year, CLSI created a table listing the most commonly isolated Staphylococcus spp. in respect to the methods used to evaluate oxacillin resistance in the introduction to Table 2C. This table was further modified to reflect species-dependent testing recommendations, with the addition of Staphylococcus epidermidis as a separate species in the M100 29th edition, and to better clarify the method options for identification of oxacillin resistance for each species. The note regarding mecC resistance was modified to indicate that MICs results for mecC are typically categorized as susceptible for cefoxitin resistant and oxacillin. In the M100 29th edition, oxacillin disk diffusion was also added as an option for the identification of oxacillin resistance of S. epidermidis isolates, although many laboratories will likely continue to use the cefoxitin disk or oxacillin MICs. Also, in Table 2C, a separate column was added for “Staphylococcus spp. indications” to define the organisms for which the breakpoints are provided, rather than having the organism names in parentheses under the listed drugs, and oxacillin disk diffusion and MIC breakpoints and cefoxitin disk diffusion have been added for S. epidermidis. Previously, the data for Staphylococcus pseudintermedius and Staphylococcus schleiferi, for which cefoxitin disk diffusion was not a viable option due to an unacceptably high error rate, were reviewed. The option to perform oxacillin disk diffusion testing was reintroduced for these species, as well. For laboratories that do not perform MALDI-TOF, once susceptibility testing, and specifically oxacillin resistance testing, is needed, laboratorians should perform appropriate testing to rule out Staphylococcus lugdunensis and perform oxacillin MIC testing on automated systems.
Clarifications A clarification was made to Table 2A, Zone Diameter and MIC Breakpoints for the Enterobacteriaceae, (p. 37) and the introduction to Tables 3B and 3C (p. 108) to indicate that additional testing, e.g., CarbaNP, mCIM, eCIM, or molecular tests, do not need to be performed once current carbapenem breakpoints have been
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implemented for the Enterobacteriaceae and P. aeruginosa unless the information will be used for epidemiological or infection control purposes. If additional testing is performed, it is also not necessary to edit interpretation results for carbapenems to “R” as long as laboratories are using the current carbapenem breakpoints. Some isolates that produce carbapenemases with a more limited spectrum of activity, such as SME or IMI, may test as susceptible to the cephalosporins. Clarifying guidance has been added to Table 2A for fosfomycin. Fosfomycin has been FDA cleared only for Escherichia coli and Enterococcus faecalis but exhibits good activity against infections by MDR Gram-negative organisms. There is great interest in fosfomycin, which can be administered in an oral sachet formulation, but clinical trials are stalled for the i.v. formulation. The previous comment “for testing and reporting of E. coli urinary tract isolates only” has been expanded to read “disk diffusion and MIC breakpoints apply only to E. coli urinary tract isolates and should not be extrapolated to other species of Enterobacteriaceae.” CLSI recommends that colonies in the disk diffusion zone should not be ignored given the absence of data suggesting clinical insignificance of these colonies.
Nomenclature Changes A couple of taxonomy updates were incorporated into the M100 29th edition. In Table 1C on what to test and report, the “Bacteroides fragilis grp.” was redefined as “Gram-negative anaerobes.” For Table 2J, Breakpoints, the “Bacteroides fragilis grp.” was redefined as “Bacteroides spp. and Parabacteroides spp.”
Intrinsic-Resistance Tables A few changes were made to Appendices B. The Citrobacter amalonaticus group was grouped with Citrobacter koseri, and Klebsiella pneumoniae, Klebsiella oxytoca, and Klebsiella variicola were grouped together. “Raoutella spp.” was added to the intrinsic-resistance table and defined as having intrinsic resistance to ampicillin and ticarcillin. Drugs previously indicated as having intrinsic resistance for the table’s A footnote were added for the Burkholderia cepacia complex.
Information that was Relocated “Using Molecular Assays for Resistance Detection” tables were previously posted on the CLSI website only. These tables have now been published in M100 29th edition as Appendix H to provide a practical approach to help laboratorians troubleshoot and resolve results discrepant between genotypic and phenotypic testing for antimicrobial resistance. Appendix H1 is focused on methicillin (oxacillin) results for S. aureus, Appendix H2 suggests strategies for vancomycin results and Enterococcus spp., and Appendix H3 pertains to extended-spectrum β-lactamases and carbapenemases for Enterobacteriaceae.
Quality Control A new quality control organism, A. baumannii NCTC 13304, was added to Table 4-A2 as a quality control (QC) strain for disk diffusion. Ranges were added for several drugs in Tables 4A-2 and 5A-2 for β-lactam combination agents, many of which are not FDA
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cleared, and for several agents that can be used for an “integrity check” of β-lactamase-producing QC strains. QC ranges were also added for N. gonorrhoeae ATCC 49226 and azithromycin in Table 4B and for P. aeruginosa and meropenem in Table 5A-1.
Summary CLSI continues to make substantial contributions to the field of AST. Several potentially impactful changes were made to the CLSI document in 2019 and clinical laboratories should be aware of the changes and incorporate them into their operating procedures. Moving forward, CLSI should aim to assist in the development of strategies for laboratories to implement the “best practice” breakpoints into their workflow. Over the past decade, CLSI has introduced several non-FDA-approved breakpoints, leading to confusion as to how to proceed, particularly in laboratories without microbiology directors. There remains significant uncertainty among laboratories regarding which antimicrobials and organisms are FDA approved. Several laboratories have only recently transitioned to current cephalosporin and carbapenem breakpoints, while others have decided not to adopt them at all. This includes the cefepime SDD breakpoints. Although the implementation of the SDD category provides promising potential benefits to clinical decision making, current CLSI breakpoints are not yet recognized by the FDA and, in the case of daptomycin for Enterococcus spp. and ceftaroline for MRSA, applies an off-label dose that may never be indicated for use by the FDA as a dosing regimen. FDA clearance is likely not possible for commercial automated AST systems, posing a challenge for breakpoint validation and implementation. Despite this, additional future non-FDA-approved breakpoints are probable as data are reassessed. The off-label use of minocycline for Stenotrophomonas infections and of amoxicillin-clavulanate for anaerobic bacteria is already under discussion. CLSI provides some recommendations through its webinars, but inclusion of detailed guidance on AST platform-specific instructions on how to transition from old breakpoints to new breakpoints and validation instructions, including calculations for categorical agreement (new SDD breakpoints will not be in categorical agreement), how to properly report results, and bridging communication between laboratory and hospital information systems and clinicians, are needed in the M100 document. In January, CLSI created a new working group that will hopefully be able to bridge some of the gaps.
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[4] Dudley MN, Ambrose PG, Bhavnani SM, Craig WA, Ferraro MJ, Jones RN, for the Antimicrobial Susceptibility Testing Subcommittee of the Clinical and Laboratory Standards Institute. Background and rationale for revised Clinical and Laboratory Standards Institute interpretive criteria (breakpoints) for Enterobacteriaceae and Pseudomonas aeruginosa. I. Cephalosporins and aztreonam. Clin Infect Dis 2013;56:1301-9. [5] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 28th informational supplement 2018; CLSI document M100-S28. Clinical and Laboratory Standards Institute, Wayne, PA. [6] Munoz-Price LS, Lolans K, Quinn JP. Emergence of resistance to daptomycin during treatment of vancomycin-resistant Enterococcus faecalis infection. Clin Infect Dis 2015;41:565-6. [7] Kelesidis T, Tewhey R, Humphries RM. Evolution of high-level daptomycin resistance in Enterococcus faecium during daptomycin therapy is associated with limited mutations in the bacterial genome. J Antimicrob Chemother 2013;68:1926-8. [8] Kelesidis T, Humphries R, Uslan DZ, Pegues D. De novo daptomycin-nonsusceptible enterococcal infections. Emerg Infect Dis 2012;18:674-6. [9] Chuang YC, Lin HY, Chen PY, Lin CY, Wang JT, Chen YC, et al. Effect of daptomycin dose on the outcome of vancomycin-resistant, daptomycin-susceptible Enterococcus faecium bacteremia. Clin Infect Dis 2017;64:1026-34. [10] Britt NS, Potter EM, Patel N, Steed ME. Comparative effectiveness and safety of standard-, medium-, and high-dose daptomycin strategies for the treatment of vancomycin-resistant enterococcal bacteremia among Veterans Affairs patients. Clin Infect Dis 2017;64:605-13. [11] Campeau SA, Schuetz AN, Kohner P, Arias CA, Hemarajata P, Bard JD, et al. Variability of daptomycin MIC values for Enterococcus faecium when measured by reference broth microdilution and gradient diffusion tests. Antimicrob Agents Chemother 2018;62:e00745-18. [12] Shukla BS, Shelburne S, Reyes K, Kamboj M, Lewis JD, Rincon SL, et al. Influence of minimum inhibitory concentration in clinical outcomes of Enterococcus faecium bacteremia treated with daptomycin: is it time to change the breakpoint? Clin Infect Dis 2016;62:1514-20.
[13] Munita JM, Panesso D, Diaz L, Tran TT, Reyes J, Wanger A, et al. Correlation between mutations in liaFSR of Enterococcus faecium and MIC of daptomycin: revisiting daptomycin breakpoints. Antimicrob Agents Chemother 2012;56:4354-9. [14] Kahlmeter G, Giske CG, Kirn TJ, Sharp SE. Point-counterpoint: difference between the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute recommendations for reporting antimicrobial susceptibility results. J Clin Microbiol 2019;57:e01129-19. [15] Drusano GL, Preston SL, Fowler C, Corrado M, Weisinger B, Kahn J. Relationship between fluoroquinolone area under the curve: minimum inhibitory concentration ratio and the probability of eradication of the infecting pathogen, in patients with nosocomial pneumonia. J Infect Dis 2004;189:1590-7. [16] Clinical and Laboratory Standards Institute. Antimicrobial Susceptibility Testing Meeting Minutes and Presentations, January 2016. [17] Tsuji M, Hackel M, Echols R, Sahm D, Yamano Y. MIC reproducibility of iron-depleted cation adjusted Mueller Hinton broth (IDCAMHB) for microdilution testing of S-649266, a novel siderophore cephalosporin, Abstr Microbe-M-470. In: Abstr 1st Microbe; 2016. [18] Public Health England. UK case of Neisseria gonorrhoeae with highlevel resistance to azithromycin and resistance to ceftriaxone acquired abroad. https://assets.publishing.service.gov.uk/government/uploads/ system/uploads/attachment_data/file/694655/hpr1118_MDRGC. pdf. [Accessed 4 October 2019]. [19] Australian Government. Department of Health. Multi-drug resistant gonorrhea. 2018. http://webarchive.nla.gov.au/gov/20181010021023/ http://www.health.gov.au/internet/main/publishing.nsf/Content/ mr-yr18-dept-dept004.htm. [Accessed 4 October 2019]. [20] Simner PJ, Bergman Y, Trejo M, Roberts AA, Marayan R, Tekle T, et al. Two-site evaluation of the colistin broth disk elution test to determine colistin in vitro activity against gram-negative bacilli. J Clin Microbiol 2019;57:e01163-18. [21] Shields RK, Clancy CJ, Pasculle AW, Press EG, Haidar G, Hao B. Verification of ceftazidime-avibactam and ceftolozane-tazobactam susceptibility testing methods against carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa. J Clin Microbiol 2018;56:e01093-17.
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