microbiologists. In this article, we will discuss some of the challenges the laboratory faces with antimicrobial susceptibility

Transcription

1 CE Update [microbiology and virology] Challenges in Antimicrobial Susceptibility Testing and Reporting Melinda D. Poulter, PhD, MT(ASCP), Janet F. Hindler, MT(ASCP) UCLA Medical Center, Department of Pathology and Laboratory Medicine, Los Angeles, CA your lab focus After reading this article, the reader should be able to discuss contemporary practices associated with antimicrobial susceptibility testing and reporting of Streptococcus pneumoniae, Staphylococcus aureus and ESBL-producing E. coli and Klebsiella sp. Microbiology exam 0203 questions and answer form are located after the Your Lab Focus section on p.885. There are unique issues associated with antimicrobial susceptibility testing and reporting of several commonly encountered bacterial pathogens. Emerging resistance may require introduction of new technology and reporting formats. Laboratory professionals should stay apprised of testing and reporting recommendations in current NCCLS standards. Accurate detection of antimicrobial resistance and effective communication of results for bacteria causing community- and hospital-acquired infections is critical to proper management of many infectious diseases. Current methods for disk diffusion and minimum inhibitory concentration (MIC) testing, which include special techniques for detecting unique resistance in some bacteria, can be found in the NCCLS standards. 1-3 To remain current with testing and reporting recommendations, laboratories must obtain the updated versions of these standards. The tables with recommendations for testing and reporting, interpreting results, and quality control are updated annually. 1 As technology allows for better understanding of antimicrobial resistance mechanisms in bacteria, new methods for in vitro detection of resistance are developed. In addition, expanded insight into the correlation of in vitro antimicrobial susceptibility testing (AST) data to clinical outcomes is changing the way AST results are reported. Consequently, there are numerous challenges for clinical microbiologists. In this article, we will discuss some of the challenges the laboratory faces with antimicrobial susceptibility testing and reporting as related to Streptococcus pneumoniae, Staphylococcus sp., and ESBL-producing E. coli and Klebsiella sp. Streptococcus pneumoniae Surveillance studies indicate that antimicrobial resistance in S. pneumoniae continues to rise, with multi-drug resistance (defined as resistance to 3 or more antimicrobials of different classes) becoming more prevalent. The incidence of penicillin resistance varies considerably around the world; however, as many as 21.5% of United States isolates are resistant to penicillin and another 12.7% have intermediate susceptibility to penicillin. 4 The number of isolates with decreased susceptibility to thirdgeneration cephalosporins is rising as well. 5 With the exception of the glycopeptides and linezolid, resistance has been reported to virtually all antimicrobial agents that would be considered for therapy of pneumococcal infections. 4-6 New recommendations for reporting of resistance in S. pneumoniae are addressed in the 2002 NCCLS standards, and clinical microbiologists should be aware of these. Testing Penicillin Resistance and 3rd-Generation Cephalosporins Penicillin resistance in S. pneumoniae is due to the presence of altered penicillin-binding proteins (PBPs), and NOT beta-lactamases. An oxacillin disk diffusion test can be used to screen for penicillin susceptibility; however, it cannot reliably detect penicillin resistance in S. pneumoniae. An oxacillin zone diameter >20 mm correlates with penicillin susceptibility, but a penicillin MIC test must be performed on isolates with zone diameters <19 mm to determine if the isolate is susceptible, intermediate, or resistant to penicillin. Although this screen is usually satisfactory for testing isolates related to nonmeningeal infections, MIC tests for penicillin and third-generation cephalosporins should be performed on cerebrospinal fluid (CSF) isolates as soon as sufficient growth is available. 1 An expeditious result is critical since the standard recommendation for treating suspected pneumococcal meningitis is cefotaxime or ceftriaxone plus vancomycin, and vancomycin treatment is often discontinued if susceptibility to an extended-spectrum cephalosporin is confirmed. 7,8 The selection of the original breakpoints for penicillin, cefotaxime, and ceftriaxone was based upon pharmacokinetic and pharmacodynamic parameters associated with CSF concentrations of these agents, as well as data defining therapeutic strategies for successful treatment of pneumococcal meningitis. Cerebrospinal fluid concentrations are lower than blood and soft tissue concentrations, which supported the lower breakpoints. However, these breakpoints are not ideal for interpreting results on isolates causing pneumonia. This led 877

2 878 Example Report for S. pneumoniae Recovered From Sites Other Than CSF S. pneumoniae (blood) MIC (µg/ml) Ceftriaxone 1.0 I (meningitis) a Ceftriaxone 1.0 S (non-meningitis) a Erythromycin >1.0 R Levofloxacin <0.5 S Penicillin b 1.0 I Trimethoprim- >4/76 R sulfamethoxazole Vancomycin 0.5 S Interpretation Report Comments: 1) Patients with meningitis require therapy with maximum doses of ceftriaxone. 2) High dose IV penicillin (eg at least 2 mil U every 4 h in adults with normal renal function) or ampicillin (eg 2 g every 6 h) are effective in treating pneumococcal pneumonia due to strains in the intermediate category. Points to Remember Regarding this Example: a Meningitis and non-meningitis breakpoints for ceftriaxone and/or cefotaxime are reported for isolates from sites other than CSF. b S. pneumoniae isolates with results in the penicillin intermediate category and causing pneumonia are often treatable with high doses of penicillin. Interpretive Criteria for Cefotaxime, Ceftriaxone, and Cefepime Susceptibility in Meningeal and Non-Meningeal Infections of S. pneumoniae MIC Interpretive Criteria Antimicrobial Agent S I R Comments T1 T2 Cefepime < >4.0 Only report interpretations for non-meningitis and include the non-meningitis notation on the report. There is no FDA-approved indication for the use of cefepime for meningitis. Cefotaxime or < >2.0 For cerebrospinal fluid (CSF) isolate, Ceftriaxone (meningitis) report only meningitis interpretations. Therapy comment: Patients with meningitis require therapy with maximum doses of cefotaxime or ceftriaxone. Cefotaxime or < >4.0 For all isolates other than those Ceftriaxone (non-meningitis) from CSF, report interpretations for both meningitis and non-meningitis. Therapy comment: For cefotaxime, use of interpretive criteria for non-meningitis requires doses appropriate for serious pneumococcal infections. Adapted from 2002 NCCLS M100-S12. Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement. intravenous penicillin (eg, at least 2 million units every 4 hours in adults with normal renal function) or ampicillin (eg, 2 g at every 6 hours) are effective in treating pneumococcal pneumonia due to strains in the intermediate category. 1 For reporting purposes, laboratories may elect to use this comment or a modification of it as shown in T1. In January 2002, NCCLS published a second set of cefotaxime and ceftriaxone breakpoints for S. pneumoniae associated with non-meningeal infections. These were given a non-meningitis designation [T2]. The older breakpoints that were based upon treating pneumococcal meningitis remain unchanged, but are now labeled as meningitis breakpoints. It is recommended that laboratories interpret results using both sets of breakpoints and list these on the patient s report, unless the S. pneumoniae isolate was recovered from CSF [T1]. For CSF isolates, only meningitis interpretations should be reported. 1 The rationale behind reporting meningitis in addition to non-meningitis interpretations on isolates from sources other than CSF is based on the fact that patients with pneumococcal meningitis may have S. pneumoniae isolated from sites other than CSF. The expanded report allows the clinician to consider these data with the patient s clinical information when making therapy decisions. Although 2 sets of breakpoints for cefepime are listed in NCCLS interpretive tables, only the nonmeningitis interpretations should be reported, since cefepime is not FDA-cleared for therapy of meningitis in the United States. Disk diffusion interpretive criteria are not available for cefepime, cefotaxime, or ceftriaxone for S. pneumoniae, as zone diameters correlate poorly with MICs for these combinations. Only drugs appropriate for the treatment of meningitis should be reported for isolates of S. pneumoniae from CSF (eg, cefotaxime or ceftriaxone, penicillin, meropenem, and vancomycin). Heffelfinger and colleagues to propose an additional set of penicillin breakpoints for isolates causing pneumococcal pneumonia, 9 but these breakpoints have not been adopted by NCCLS. Alternatively, NCCLS suggests a comment be added to patient reports when penicillin-intermediate results (MIC µg/ml) are obtained for S. pneumoniae isolated from sources associated with pneumonia. The NCCLS comment reads High doses of Testing and Reporting Macrolides and Fluoroquinolones The 2 most common mechanisms causing macrolide resistance in S. pneumoniae are target alteration and active

3 Abstract Table Isolate Antimicrobial Agent Points to Remember S. pneumoniae Penicillin Report penicillin as susceptible when oxacillin zone diameter is >20 mm. Perform penicillin (and cefotaxime or ceftriaxone) MIC on isolates with oxacillin zone diameter <19 mm. Do not perform oxacillin screen on CSF isolates. Perform MIC ASAP. Add NCCLS comment to report when penicillin intermediate results are obtained from isolates from sources other than CSF. efflux. 4 Target alteration is usually due to ermb-encoded erythromycin ribosome methylase, which confers the MLS B phenotype. Isolates of the MLS B phenotype are resistant to the macrolides (eg, erythromycin), lincosamides (eg, clindamycin), and streptogramins B (an agent not available in the United States). 10 It is very unusual for isolates with ermb to demonstrate in vitro resistance to erythromycin only. Resistance to macrolides alone (M phenotype) is more commonly due to expression of a mefaor mefe-encoded efflux pump, which Cefotaxime and ceftriaxone General transports the drug out of the cell before it reaches its target. 10 The newer quinolones such as levofloxacin, gatifloxacin, and moxifloxacin show increased activity against S. pneumoniae as compared to ciprofloxacin. Although active efflux may result in low-level resistance to the fluoroquinolones, target modification is the primary mechanism of fluoroquinolone resistance in S. pneumoniae. 11 The targets appear to be genes coding for enzymes involved in DNA synthesis, most frequently parc and gyra. The development of resistance is a Perform cefotaxime or ceftriaxone MIC ASAP on CSF isolates. Report "meningitis" and "non-meningitis" interpretations for isolates from sources other than CSF. Report "meningitis" interpretations only for isolates from CSF. Cannot be reliably tested by disk diffusion. Report only drugs appropriate for treatment of meningitis on isolates from CSF. Staphylococci Oxacillin Report oxacillin-resistant staphylococci as resistant to all beta-lactams. Macrolides (eg, erythromycin) and lincosamides (eg, clindamycin) Vancomycin Erythromycin resistant and clindamycin susceptible isolates may exhibit inducible clindamycin resistance. Consider "D test" for verification. Report all suspected VISA or VRSA isolates immediately to local public health department and send isolate for confirmation. Klebsiella spp., E. coli Screening tests Use ESBL-specific breakpoints. cefpodoxime, ceftazidime, aztreonam, Sensitivity of ESBL detection is increased cefotaxime, ceftriaxone if more than one agent is tested. Confirmatory testing cefotaxime and ceftazidime with and without clavulanic acid If ESBL identified, all cephalosporins, penicillins and aztreonam reported as resistant. ESBL testing may not be necessary on urine isolates from patients with acute, uncomplicated cystitis. 2-step process in which a mutation occurs in the 1 target, causing a reduction in activity of the antimicrobial agent. This is followed by mutation of a secondary target, that causes an additional decrease in activity. 12 Isolates harboring a single mutation in either parc or gyra may have elevated MICs to some fluoroquinolones, but can remain in the susceptible to intermediate range, while isolates with multiple mutations are typically resistant to all the fluoroquinolones. 11,13 Because isolates with single step mutations may be more prone to developing resistance during 879

4 880 therapy, as compared to fully susceptible isolates with very low MICs, the ability to detect elevated MICs, even if the isolate is still susceptible, may be clinically useful. 13 NCCLS standards list gatifloxacin, levofloxacin, moxifloxacin, and sparfloxacin together in a single box, separated by or, meaning there is nearly complete cross susceptibility and cross resistance among the 4 agents for S. pneumoniae. 1 The same principle applies to S. pneumoniae and with respect to erythromycin, azithromycin, or clarithromycin. For example, laboratories may choose to test only erythromycin and add a report comment stating that an erythromycin-resistant isolate is resistant to azithromycin and clarithromycin. Susceptible results can be similarly reported with an appropriate comment. Staphylococci Increasing antimicrobial resistance in staphylococci has been a concern since the early reports of oxacillin resistance in Staphylococcus aureus. 14 Historically, oxacillin-resistant S. aureus (ORSA) were referred to as MRSA or methicillin-resistant S. aureus, when both oxacillin and methicillin were available for in vitro testing. Because of its superior performance with in vitro test systems, oxacillin is now the preferred agent for testing and the worldwide availability of methicillin is limited. However, the term MRSA continues to be used in the medical literature to describe S. aureus that are resistant to the penicillinase-stable penicillins, including oxacillin, methicillin, nafcillin, and dicloxacillin. In addition to understanding how to detect and report oxacillin resistance, it is important for clinical laboratories to be aware of inducible clindamycin resistance and newer resistance mechanisms such as those seen in VISA and VRSA. Confirming and Reporting Oxacillin Resistance Methicillin-resistant S. aureus is most commonly associated with nosocomial infections and is typically resistant to multiple antimicrobial agents. However, MRSA associated with community-acquired infections is often susceptible to non-beta-lactam agents. 15 Oxacillin inhibits cell wall synthesis in staphylococci by binding to penicillin binding proteins, enzymes necessary for cell wall synthesis. This binding interferes with the production of peptidoglycan, which results in weakening of the cell wall and cell lysis. 16 Oxacillin resistance in both S. aureus and coagulasenegative staphylococci (CoNS) is due to a chromosomal gene, meca, which codes for a variant of the penicillin binding protein PBP2. This altered PBP, designated PBP2a (or PBP2), performs functions similar to that of PBP2 during cell wall synthesis. However, oxacillin binds poorly to and does not interfere with PBP2a. 17 There are several tests for the detection of oxacillin resistance in staphylococci. Oxacillin-salt agar screening plates can be used for S. aureus, but are unreliable for CoNS. Disk diffusion and broth or agar dilution methods can be used for all staphylococci, although the zone diameter and MIC interpretive criteria for S. aureus and CoNS are different. 1 Slight modifications (eg, extended incubation, addition of NaCl to MIC tests) to standard disk diffusion and MIC methods are required to ensure detection of heteroresistant populations (populations of meca containing cells, some of which express susceptibility and others that express resistance to oxacillin). Because the presence of PBP2a confers resistance to all beta-lactams, oxacillinresistant staphylococci must be reported as resistant to all beta-lactam agents, including penicillins, cephalosporins, beta-lactam/beta-lactam inhibitor combinations, and carbapenems, even if in vitro testing of these agents demonstrates susceptibility. 1 The ability of commercial systems to detect oxacillin resistance in staphylococci is well documented. 18,19 Some laboratories that use a commercial system for S. aureus inoculate an oxacillin-salt agar screening plate concurrently to increase the likelihood that MRSA will be identified. However, once a clinical laboratory verifies satisfactory test performance of the commercial system in the hands of their laboratory personnel, the need to perform parallel testing with the oxacillin-salt agar screen test on every S. aureus is questionable. Rapid screening methods are now available for detection of meca or PBP2a. Methods based on PCR that target meca are the gold standard by which new methods are measured ; however, the use of PCR may not be feasible in most clinical laboratories. A new latex agglutination test for detection of PBP2a in S. aureus and CoNS has recently been FDA cleared. This method offers a high level of sensitivity (>97%, in most cases) and approaches 100% specificity with both S. aureus and CoNS The rate of false negatives reported in initial experiments using the PBP2a assay for S. aureus and CoNS varies varied from 0% to 5%, and in most reports were thought to be due to the presence of heteroresistant populations that decreased the amount of PBP2a available for binding. Most discrepancies occurring in both S. aureus and CoNS were resolved upon repeat testing with a larger inoculum, incubation for 10 minutes instead of the 3 minutes suggested by the manufacturer, or induction with an oxacillin disk prior to testing. 18,20-22 The current package insert states that isolates of S. aureus can be tested directly from growth on primary culture plates or subcultures, but a heavy inoculum consisting of approximately 1.5 x 10 9 cells in a 5 µl loop is necessary. Subculture and induction with an oxacillin disk to produce adequate PBP2a is required for testing of CoNS. The manufacturer makes no suggestions for extended incubation beyond 3 minutes. In most instances, tests for meca or PBP2a provide results more rapidly than conventional susceptibility testing methods. According to NCCLS, staphylococci that test positive for meca or PBP2a should be reported as oxacillin resistant. 1 Resistance and Decreased Susceptibility to Vancomycin Vancomycin, a glycopeptide, has been the treatment of choice for serious S. aureus infections due to oxacillinresistant strains or for patients known to

5 be penicillin-allergic. The first isolate demonstrating intermediate resistance to glycopeptides was recovered in Japan in 1996, with subsequent reports of vancomycin-intermediate S. aureus (VISA) from the United States and Europe. 23,24 These isolates demonstrated vancomycin MICs of 8 µg/ml and resistance to teicoplanin, a glycopeptide that is currently unavailable in the United States. Most VISAs are MRSAs, although at least 1 oxacillin-susceptible VISA has been reported. 25 Vancomycinresistant strains of CoNS strains have also been noted Of great concern from a clinical and public health perspective is the recent isolation of the first MRSA demonstrating high-level resistance to vancomycin. 29 The mechanism of decreased susceptibility to vancomycin in VISA is unrelated to that found in vancomycinresistant enterococci. 23,25,30 Broth microdilution or Etest can detect VISA; however, disk diffusion is unreliable. 23,30 A commercial BHI agar screen plate (BHI-V) used for the detection of vancomycin-resistant enterococci has also proven useful in the detection of VISA. Limited studies indicate that some automated methods can reliably detect VISA. 23,30 However, the current CDC guidelines ( ncidod/hip/aresist/search.htm) state that the isolate should be tested on 2 separate occasions using an acceptable broth microdilution method, Etest, or agar dilution method using a full 24- hour incubation. The laboratory should reconfirm the genus and species identification and ensure that the strain is in pure culture when performing susceptibility testing. Strains demonstrating an MIC >4 µg/ml should be considered as presumptive Staphylococcus aureus with reduced vancomycin susceptibility. The first clinical isolate of MRSA with high-level vancomycin resistant resistance (MIC >32 µg/ml (VRSA)) was detected using a commercial MIC testing system. Testing at the CDC verified a MIC >128 µg/ml to vancomycin and identified the presence of a vana genetic determinant, believed to be transferred from a co-existing vancomycin-resistant Enterococcus faecalis. 29 All suspicious or confirmed isolates of VISA or VRSA should be reported immediately to the institution s infection control department as well as the state and local health departments and CDC, with the isolate saved and expeditiously sent to the local public health department for confirmation. 31 Example report for S. aureus S. aureus MIC (µg/ml) Erythromycin a >8.0 R Oxacillin b >4 R Penicillin >0.25 R Vancomycin 0.5 S [I1] D" test indicating the presence of inducible clindamycin resistance in an isolate of S. aureus. Inducible Resistance to Clindamycin Resistance to macrolides, lincosamides, and streptogramin B in staphylococci is most frequently due to methylation of 23s rrna by erma or ermc-encoded methylases, resulting in decreased binding of the drug to its rrna target. 10 If the erma or ermc are continuously expressed, erythromycin, clindamycin, and other members of the MLS B group will exhibit resistance. However, the genes sometimes require the presence of an inducing agent, such as erythromycin, to express resistance to clindamycin and other members of the MLS B group. 10,32 These isolates will show in vitro resistance to erythromycin but susceptibility to clindamycin because clindamycin does not act as an inducing agent. In order for the inducible phenotypeclindamycin resistance to be demonstrated, a special test ( D test) must can be performed. Using standard disk diffusion procedures, an erythromycin disk is placed near a clindamycin disk (15 mm from center to center). Erythromycin induces production of the methylase, which allows clindamycin resistance to be expressed, but only within the area where both erythromycin and clindamycin molecules coexist. This causes the appearance of a D-shaped zone around the clindamycin disk (I1). 10 It is unclear whether inducible clindamycin resistance is significant in vivo, particularly if the patient has not been exposed to erythromycin or another macrolide. 10 NCCLS does not currently address this issue. There are several options for reporting antimicrobial susceptibility testing results of erythromycin-resistant, clindamycinsusceptible staphylococci: 1) change clindamycin results to resistant; 2) report clindamycin following performance of the induction test; or 3) refrain from reporting clindamycin and perform the induction test by special request only (T3). If clindamycin resistance is only apparent after induction, a comment to this effect can be added to the report. If Interpretation T3 Report Comments: 1) Oxacillin resistant staphylococci are resistant to cefazolin and all other beta-lactams. 2) Contact laboratory if clindamycin results are needed. Points to remember regarding this example: a Clindamycin results were not reported because the organism tests erythromycin resistant and clindamycin susceptible. Testing for inducible clindamycin resistance is performed upon physician request. b Oxacillin-resistant staphylococci are considered resistant to all penicillins, carbapenems, cephems, and beta-lactam/beta lactamase inhibitor combinations, regardless of in vitro susceptibility. 881

6 882 Example of Preliminary and Final Reporting Formats for Isolates Screened and Confirmed as ESBL Producers PRELIMINARY REPORT K. pneumoniae (blood) MIC (µg/ml) Ampicillin >32 R Cefazolin >32 R Cefoxitin 1.0 S Ciprofloxacin 0.5 S Gentamicin a >16 R Imipenem <0.25 S Piperacillin-tazobactam 16/4 S Trimethoprim- >4/76 R sulfamethoxazole a no induction is demonstrated (no D zone), the isolate should be reported as clindamycin susceptible. In this case, erythromycin resistance is likely due to a less common mechanism, such as the presence of macrolide efflux pumps (encoded by msra or msrb). 10,32 Extended-Spectrum Beta- Lactamases (ESBLs) Extended-spectrum beta-lactamases (ESBLs) are beta-lactamases that mediate resistance to extended-spectrum cephalosporins, (eg, cefotaxime, ceftriaxone, and ceftazidime), monobactams (eg, aztreonam), and extended-spectrum Interpretation Report Comment: This K. pneumoniae is suspicious for extended-spectrum beta-lactamase (ESBL) production; confirmatory tests pending. b Points to remember regarding this example: a ESBL-producing organisms are often resistant to multiple classes of antimicrobial agents. b When ESBL screening tests suggest the possibility of an ESBL-producing organism, results of cephalosporins, penicillins, and aztreonam are withheld (if in vitro results are susceptible or intermediate) pending confirmatory tests. For example: hold results of cefotaxime MIC of 8.0 µg/ml. FINAL REPORT K. pneumoniae (blood) MIC (µg/ml) Ampicillin >32 R Cefazolin >32 R Cefoxitin 1.0 S Cefotaxime c - R Ciprofloxacin 0.5 S Gentamicin >16 R Imipenem <0.25 S Piperacillin-tazobactam 16/4 S Trimethoprim- >4/76 R sulfamethoxazole Interpretation T4 Report Comment: Confirmatory tests of this K. pneumoniae indicate unusual resistance [extended-spectrum beta-lactamase (ESBL) production]; Infectious Disease consult suggested. c Points to remember regarding this example: c When ESBL confirmatory tests indicate the isolate is an ESBL-producer, all cephalosporins, penicillins, and aztreonam must be reported as resistant, regardless of in vitro susceptibility testing results. penicillins (eg, ticarcillin and piperacillin). They are most commonly found in E. coli, K. pneumoniae, and K. oxytoca. 33 Their true incidence is difficult to discern due to differences in testing and reporting practices, but marked differences have been reported based upon geographic location. 34 Reports of treatment failures, due to use of aztreonam extended-spectrum cephalosporins prescribed for serious infections such as bacteremia or meningitis that were caused by an ESBL-producing organism, have drawn attention to the need for ESBL detection and reporting protocols in clinical laboratories Testing and Reporting ESBLs Extended-spectrum beta-lactamases arise from stepwise mutations in plasmid-borne genes that encode common beta-lactamases (eg, TEM and SHV) in gram-negative organisms such as E. coli and K. pneumoniae. They confer variable levels of resistance to extendedspectrum cephalosporins (eg, cefpodoxime, ceftazidime, cefotaxime, ceftriaxone) and monobactams (eg, aztreonam), but generally have no effect on cephamycins (eg, cefoxitin) or carbapenems (eg, imipenem or meropenem). Beta-lactamase inhibitors (eg, clavulanate, sulbactam, tazobactam) usually block the activity of ESBLs, and this feature is the basis for in vitro confirmatory tests for ESBLs. Plasmids that carry ESBLs genes often have multiple genes encoding antimicrobial resistance to agents of other drug classes. Therefore, ESBL-producing bacteria are often, but not always, resistant to multiple antimicrobial agents. 33 Although ESBLs may be present in several members of the Enterobacteriaceae, NCCLS has standardized testing recommendations for E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca only. Specific breakpoints for ESBL screening in these organisms have been developed for cefpodoxime, ceftazidime, aztreonam, cefotaxime, and ceftriaxone. Cefpodoxime offers the highest level of sensitivity for ESBL detection, and recent modifications to the NCCLS screening breakpoints have increased the specificity of this test. 1 However, the overall sensitivity of detection can be increased if more than 1 agent is utilized. 1,39 Isolates that show a positive screen test should be subjected to confirmatory testing and any susceptible result for extended-spectrum cephalosporins, penicillins, and aztreonam should be withheld until confirmatory testing is complete. 1 An explanatory comment should be added to the preliminary report as seen in T4. Testing of cefotaxime and ceftazidime, with and without clavulanate, is used for ESBL confirmatory testing. Clavulanic acid restores the activity of cefotaxime and/or ceftazidime

7 against ESBL-producing strains (T4). 1 Etest strips (AB Biodisk M, Solna, Sweden) and disks containing cefotaxime or ceftazidime with clavulanate are commercially available for ESBL confirmation tests (Oxoid M, Ogdensburg, NY; Mast Group Ltd M, Mercyside, UK; Becton Dickinson Microbiology Systems M, Cockeysville, MD). Vitek (biomerieux M, Durham, NC) and Microscan MicroScan (Dade MicroScan M, Sacramento, CA) automated platforms also offer a FDA-cleared ESBL confirmatory test. Some Vitek GNS cards used for routine susceptibility testing include an ESBL confirmatory test, which precludes the need for initial ESBL screening tests. For isolates confirmed as ESBL producers, all cephalosporins, penicillins, and aztreonam are reported as resistant, regardless of in vitro susceptibility testing results. However, cephamycin and carbapenem in vitro AST results are reported as tested (T4). 1 If the isolate cannot be confirmed as an ESBL producer, suggesting an alternative resistance mechanism, the results are reported according to the original interpretation. A comment in the final report should clarify that additional testing did not confirm the presence of an ESBL-producing organism. Issues Related to Site of Specimen Collection Testing for the presence of ESBLs in isolates recovered from patients with bacteremia or other serious infection is highly recommended. However, NCCLS states that the decision to test for ESBL production in all urine isolates of E. coli and Klebsiella spp. should be made on an institutional basis. 1 This suggestion is based on the fact that acute, uncomplicated cystitis caused by ESBL producers is likely to respond to cephalosporins, penicillins, or aztreonam because high levels of the drug can be achieved at the site of infection. 40 The decision to test for ESBL-producing E. coli is reported from a body site other than urine, but the E. coli isolated from urine is not reported as an ESBL. This implies the presence of 2 different organisms, when in fact they may be the same. In addition, the infection control staff may want to know of all ESBLs, to allow them to impose appropriate infection control measures. If it is known with certainty that the organism was isolated from an outpatient with acute uncomplicated cystitis, testing for ESBL production might be unnecessary. Because this information is often difficult for the laboratory to obtain, it is unrealistic for most institutions to refrain from ESBL testing on selected urine isolates. Hidden Challenges Facing Clinical Laboratories The ability of the clinical laboratory to determine the extent to which an isolate is susceptible to an antimicrobial agent offers some assurance to the clinician that the agent selected will be effective. Therefore, laboratory professionals must use accepted methods for bacterial identification as well as AST. This requires access to current NCCLS AST standards and continuing education programs that alert microbiologists to potential changes in testing and reporting recommendations. A consistent flow of information between the laboratory and infectious disease service, pharmacy, and infection control department is equally important to ensure that the appropriate agents are selected for testing, the appropriate level of microbial identification and susceptibility testing is performed, and that the data generated is conveyed to the clinician rapidly and reliably. All of these factors come together to ensure that AST results are useful to the clinician. Given this as a primary goal of AST, and taking into account the emergence of new mechanisms of resistance, accurate detection of resistance and appropriate reporting of AST results is of paramount importance in patient care. 1. NCCLS. Performance standards for antimicrobial susceptibility testing; twelfth informational supplement. M100-S12. Wayne, PA: NCCLS; NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved Standard M7-A5. Wayne, PA: NCCLS; NCCLS. Performance standards for antimicrobial disk susceptibility tests, 7th ed. Approved Standard, M2-A7. Wayne, PA: NCCLS; Doern GV, Heilmann KP, Huynh HK, et al. Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during , including a comparison of resistance rates since Antimicrob Agents Chemother. 2001;45: Whitney CG, Farley MM, Hadler J, et al. Increasing prevalence of multidrug-resistant Streptococcus pneumoniae in the United States. N Engl J Med. 2000;343: Thornsberry C, Sahm DF, Kelly LJ, et al. Regional trends in antimicrobial resistance among clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the United States: Results from the TRUST Surveillance Program, Clin Infect Dis. 2002;34:S4-S The choice of antibacterial drugs. In: Abramowicz M, editor. The Medical Letter. New Rochelle, NY; 2001: Sanford JP, Gilbert DN, Moellering RC, Jr., et al. The Sanford Guide to Antimicrobial Therapy. 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