Controversial Issues in Susceptibility Testing: Point/Counterpoint. April N. Abbott, PhD D(ABMM) Romney M. Humphries, PhD D(ABMM)

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1 Controversial Issues in Susceptibility Testing: Point/Counterpoint April N. Abbott, PhD D(ABMM) Romney M. Humphries, PhD D(ABMM)

2 Disclosures RMH: Funding from BD, biomerieux, Beckman-Coulter, GenMark, BioFire, Accelerate Diagnostics, Merck, Allergan Consulting for DNAe, SlipChip, Luminex, Merck, Allergan, Shionogi, biomerieux, Zavante, MicrobeDx Employment: Accelerate Diagnostics (as of October 29 th )

3 Case 1: ESBL or not to ESBL? 8 year old girl, complex urological history with several reconstructive surgical procedures Presents with E. coli in the blood, urine, with following profile: Team: This smells like an ESBL can you test for us? Antimicrobial MIC (μg/ml) Amikacin 2 S Ampicillin >32 R Cefazolin >32 R Cefepime 4 S-DD Ceftazidime 0.5 S Ceftriaxone >32 R Ciprofloxacin >2 R Ertapenem <0.25 S Gentamicin 0.5 S Piperacillin-Tazobactam 16 S Tobramycin 1 S Trimethoprim-Sulfa >4 R

4 Why did CLSI get rid of the ESBL test? New drug (eg: cephalosporin) introduced to market in context of no resistance Emergence of a new β-lactamase (e.g., ESBL) Old Paradigm Screen isolates for resistance mechanism (elevated MICs near S breakpoint are suspicious ) Perform special confirmatory test for resistance mechanism Change the susceptibility report for all drugs of that class, if resistance mechanism detected

5 What exactly is a clinical breakpoint? Cut-off that identifies a patient, by MIC, for which there is a high probability of clinical response to a properly dosed antimicrobial. Previous breakpoints established over 20 years ago (before ESBLs) Increased knowledge of beta-lactam resistance mechanisms Many types of beta-lactamases (ESBLs, ampcs, other) CLSI ESBL test only standardized for E. coli, Klebsiella spp. and Proteus mirabilis (mid 1990s) ESBLs occur in other species ESBLs not detected if multiple R mechanisms in an isolate Note: in 2010, among US isolates with MIC in new resistant range (>2) only 50% were E. coli, Kleb or P. mirab

6 Clinical Data Human Data: suboptimal responses to 3rd generation cephalosporins with MICs of 2, 4, or 8 g/ml *many studies in literature that show poor response for ESBL irrespective of MIC, had suboptimal dosing 42 patients, Andes and Craig MIC % Response <= >=8 11

7 Resistance mechanism does not matter in this! Cephem (neutropenic mouse thigh model) For ESBL positive isolates: Low cephem MICs due to being poor substrates for specific ESBL (eg: CTX-M-1 does not hydrolyze ceftazidime) Animal studies indicated % T > MIC did not differ for ESBL positive vs. ESBL negative Enterobacteriaceae (i.e. same exposure-response curve) All about the MIC Ambrose et al. Abstract A th ICAAC Washington DC 2004

8 Bottom line 1) ESBL test was a band-aid 2) ESBL test ONLY works for E. coli, Klebsiella spp., P. mirabilis 3) ESBL test unnecessarily eliminates some cephems from consideration 4) ESBL test does not predict clinical outcome, MIC does but you want to use ESBL test for epidemiological purposes? GO FOR IT! CLSI stills endorses it s use for this purpose

9 But, what about this Bacteremic patients receiving empiric pip/tazo have a greater likelihood of death compared to patients that got a carbapenem Tamma, 2015, CID, 60(9)

10 Source and MIC may play a role No patients with UTI as the source of bacteremia died as compared to significant mortality when bacteremia was attributed to a nonurinary source Patients receiving pip/tazo with low MIC (2 µg/ml) faired well; however MICs above 2 were associated with mortality Retamar, AAC, 2013, 57(7)

11 What about other agents? Wang et. al. found that cefepime empiric treatment of an ESBL was associated with 2.9 times greater risk of death compared to therapy with a carbapenem Lee et. al. demonstrated that mortality increases with the cefepime MIC Wang, Open Forum Infect Dis, 2016, 3(3) Lee, 2013, CID, 56(4)

12 Bottom, bottom line We still don t know. Data are still being gathered because many labs were late to adopt the 2010 breakpoints For UTIs (and bacteremia secondary to a UTI) caused by an ESBL + organism, treatment with a susceptible beta lactam, such as pip/tazo, seems to be a reasonable approach Empiric therapy with a non-carbapenem may be risky Inoculum effect has been raised as a possible reason for treatment failure with certain beta-lactam antibiotics To agree with Dr. Humphries MIC matters

13 Case 2: More mechanism shenanigans 62 year old woman with advanced pancreatic cancer Vomiting & fever after surgery CT scan: fluid collection in liver, inflammatory ascites Blood cultures: Gram negative rods ID: K. pneumoniae AST: pretty darn R Is this a carbapenemase producer? Ertapenem Meropenem Imipenem Cefepime Ceftriaxone 4 ug/ml, R 1 ug/ml, S 8 ug/ml, R 16 ug/ml, R 16 ug/ml, R Ciprofloxacin 1 ug/ml, S SXT <=2 ug/ml, S

14 Is testing for carbapenemase necessary? CLSI Guidance (M100S, 27 th Edition) Key point: Such testing is not currently recommended for routine use PROVIDED using post-2010 breakpoints!

15 % of 208 isolates First off PLEASE, adopt the current BPs! It s the BEST way to detect clinically meaningful carbapenem resistance! 25 S19 S27 The old breakpoints 20 MISS many KPC producers!! S to ERT S to IMP S to MER S to all 3 S to IMP/MER Even worse for other carbapenemases Data: UCLA, n=208 isolates

16 Impact of delay Computer simulation data for Orange County, CA 5 year delay in implementing breakpoints = 8, 500 more CRE carriers in one county

17 Need help with this? CLSI AST News Update

18 Like for ESBLs, clinical outcome correlates with MIC, not carbapenemase presence 162 patients with K. pneumoniae BSI 95 VIM-negative 53 VIM-positive/carbapenem-S 14 VIM-positive/carbapenem R ESBL production, HR= 1.02 ( ) p=0.95 VIM-1 production, HR = 1.67 ( ) p=0.2 Carbapenem MIC >4 ug/ml HR=3.69 ( ), p=0.02 Daikos 2009 AAC 53:

19 Carbapenemase test options CLSI Methods FDA Cleared Modified Hodge Test CarbaNP mcim Xpert CarbaR Easy Difficult Easy Easy Good for KPC K. pneumoniae Good for KPC, MBL Good for almost all Good for all tested Poor for NDM Poor for OXA-48-like? $$ OBSOLETE AS OF 2018!! Can your lab do the verification? Bring on new test? How many carbapenem-r isolates do you have? Is it wort the squeeze?

20 Example: carbapenemase testing challenges UCLA, 2014: is this a carbapenemase producer? Amikacin Gentamicin Tobramycin Aztreonam, Cefepime, Cetazidime, Ceftriaxone Ertapenem Imipenem Meropenem Ciprofloxacin Levofloxacin >32 >10 >10 >32 >32 >32 >32 >8 2 2 >2 >8 R R R R R R R R I I R R Piper-tazo >128 R - + iso1 iso2 Carba-NP negative MHT positive KPC gene, negative IMP gene, negative VIM gene, negative SME gene, negative Oxa-48 gene, negative NDM gene, negative WGS: Oxa-232 New carbapenemase in US Trim-sulfa >4/80 R

21 Carbapenemase testing, yes please! Introduction of new methods and FDA-approved tests has changed the game

22 Carbapenemase testing impact on patient outcome Use new breakpoints (and dodge the Hodge ) Susceptibility data is likely the best indicator of success, however Not all carbapenemase + organisms are resistant to all the carbapenems using the current breakpoints KPC and OXA-48 isolates that are susceptible to imipenem Outcome studies are challenging given the relatively low prevalence of carbapenemase-producing Enterobacteriaceae, the use of combination therapy, and limited alternative treatment options

23 Know your AST system Acinetobacter vs imipenem CLSI = old CLSI breakpoints EUCAST = current CLSI breakpoints VME = actually resistant but the system reports the organism as susceptible Reported VMEs are less frequent with Enterobacteriaceae, but still may be an issue REMEMBER imipenem is labile which may contribute to ME Markelz, AAC, 2011, 55(10)

24 Let s talk about epidemiology April: I have a lot of epi-type slides for a good reason to do it do you want these? Yes please! I ll put in a few slides if you can dump yours in to round it out. I will be able to get the gist from your slide.

25 Why does carbapenem resistance mechanism matter? CRE phenotype may be due to: Carbapenemase production ( CP ) OR Porin mutation + other beta-lactamases 25

26 Spotting a C pase producer is hard (impossible)! ploads/attachment_data/file/344071/p_8i1.1.pdf 26

27 Why are carbapenemase producers epidemiologically important? Current CRE epidemic is due to carbapenemase producers USA: KPC harboring ST258 strain of K. pneumoniae Europe: OXA-48-like, NDM Carbapenemase genes are on highly mobile plasmids Transmitted by movement of patients colonized / infected with CP-CRE 27

28 1 Year of KPC Transmission Across 4 Counties 24 / 40 CRE treated at LTACH-A Patients transferred extensively across 26 facilities KPC confused with ESBL by epidemiologists Outbreak unhindered for 5 months Wi et al Clin Infect Dis. 53:

29 KPC (Klebsiella pneumoniae Carbapenemase) First report 1997 North Carolina High level of enzyme typically produced Mostly K. pneumoniae, but also other GNR Plasmid with KPC gene generally has other R genes including ESBLs

30 Metallo β-lactamase (MBL) Type of Carbapenemase Zinc required for activity NDM (New Delhi MBL) most common worldwide; India and Pakistan First report 2008 in Swedish patient hospitalized in India VIM (Verona integron-encoded MBL) Europe, SE Asia IMP (Imipenemase) Europe

31 NDM IMP 72 NDM in IL ERCP outbreak 1 VIM 6 IMP in CA Stanford outbreak 2 8 VIM in KY NICU outbreak 3 1. MMWR (51); JCM : MMWR (7);190

32 Medical Tourism

33 OXA Carbapenemases First described in Acinetobacter baumannii in 1985 OXA-48 Commonly found in Europe and Africa; rare in USA First report 2008 in Turkey Mostly K. pneumoniae, E. coli Variants include OXA-181 and OXA-232 OXA-48-like Weakly hydrolyze carbapenems & cephalosporins (need porin defect and presence of ESBL for full CRE phenotype)

34 OXA-48-like Carbapenemases

35 Case 2 continued: Should all drugs be tested in-house? So remember that patient? Can you test: - Fosfomycin - Colistin - Ceftazidime-avibactam

36 Guidance from CLSI and CAP CLSI, M100S 27 th Edition Instructions for Use: each laboratory should develop a protocol to address isolates that are confirmed as resistant to all agents on their routine test panels. This protocol should include options for testing additional agents in-house or sending the isolate to a reference laboratory. CAP- MIC.21944: There are written policies for testing supplemental agents when needed on isolates resistant to routinely tested antimicrobial agents.

37 In case you ve been living under a rock

38 no CLSI breakpoints in M100S 27 th Ed? Pharma proposes breakpoint in new drug application (NDA) to FDA FDA reviews & approves (or requests changes) FDA ONLY approves BP for bugs with clinical indications for use of the drug i.e.: infections seen in clinical trial Approved breakpoint listed in drug package insert Pharma comes to CLSI to propose breakpoint (OPTIONAL) CLSI reviews & approves FDA breakpoint OR defers for 2 years If pharma doesn t come to CLSI, no breakpoints will be listed in M100S (e.g., tigecycline) Labs can use FDA or CLSI BPs AST manufacturers must use FDA BPs

39 Where to find the FDA breakpoints in drug package insert Microbiology: Example: Ceftazidimeavibactam Package Insert

40 Supplemental Drugs to Consider for AST of MDR GNR Automated AST? Disk? Gradient Strip? Sensititre Colistin (polymyxin B) No No! No! RUO Fosfomycin No Yes, E.coli RUO No Ceftazidime-avibactam Soon Yes Soon Yes Ceftolozane-tazobactam Soon Yes Liofilchem Yes

41 Current timelines for new drug AST development/clearance Vitek 2 Phoenix MicroScan Time to clearance Etest Trek BD DISK Hardy Disk Note: time to market may be longer Months Data derived from FDA website clearance dates for drug X

42 Bottom line TALK to your clinical colleagues! Have them prioritize for you what drugs are needed vs. wanted Evaluate how often do you encounter CRE at your institution? MDR P. aeruginosa? Is the drug(s) on formulary? What are your testing options? (hint: it won t be your automated system)

43 Case 3: Scoop on poop 65 year old man, MSM Presents to ED, febrile & dehydrated 10 days of non-bloody diarrhea PMH: HIV, non-traumatic tendon rupture in days cefixime, no improvement Stool PCR: Shigella Reflex culture (required in some states): Shigella sonnei SHOULD WE DO AST ON THIS ORGANISM?

44 Is diarrhea treated with antibiotics? ALWAYS Treat Typhoidal Salmonella Shigella (population dependent) MAYBE Treat Campylobacter Salmonella Vibrio NEVER Treat STEC

45 Challenges Need to maintain two systems (molecular and culture) which adds cost Most states require a submission of the isolate OR the sample for some, but not all GI bugs, which gives laboratories a reason to abandon culture-based methods Certain pathogens require special conditions (e.g. Campylobacter and some Vibrio) AST device manufacturers make it difficult

46 Current CLSI recommendation M100: When fecal isolates of Salmonella and Shigella spp. are tested, only ampicillin, a fluoroquinolone, and trimethoprim-sulfamethoxazole should be reported routinely. Typhoidal Salmonella and Shigella from intestinal sources should get AST.

47 Key AST challenges (Salmonella and Shigella) Antimicrobial Agent Ciprofloxacin Levofloxacin Azithromycin Issue Few manufacturers have dilutions low enough to report susceptibility Not all mechanisms are detected without low breakpoints Few manufacturers have dilutions low enough to report susceptibility No disk diffusion breakpoints No approved breakpoints If not testing, consider producing and antibiogram based on data received from reference or public health laboratory to watch for emergence of resistance

48 Are the empirical drugs predictably active? FQs SXT Azithro Tet Typhoid Salmonella Shigella Campylobacter Typhoid salmonella Shigella Campylobacter Salmonella Salmonella Typhoid Salmonella Shigella Campylobacter Shigella Campylobacter Green, <5% resistance per CDC 2014 data on human isolates; red: <90% susceptibility

49 Resistance is regional Shigella resistance rates in USA, 2015 SXT FQs Azithro Cephalothin

50 Resistance is patient-specific

51 UCLA policy UCLA Policy: - Perform PCR for Salmonella, Shigella, Campylobacter and STEC - Attempt to recover, in culture, routine, Salmonella & Shigella - Routine AST on Shigella. Routine on Salmonella if infant - All others: on request only

52 Shigella and fluoroquinolones Any patient with a Shigella infection could carry a strain harboring a quinolone resistance gene with a ciprofloxacin MIC of μg/ml.

53 Salmonella challenges & solutions Challenge Commercial system does not have cipro / levo concentrations low enough for new BP Possible Solution - Use DD for ciprofloxacin - Use Etest (verify?) for cipro/levo No levofloxacin disk breakpoints - Use Etest (verify?) - Use proposed BPs and disclaim 1,2 Pefloxacin not available - Test and report cipro disk 1. Deak et al JCM : Sjolund-Karlsson et al 2014 JCM 52:877

54 FQ Etest Etest not FDA-cleared with CLSI BPs Appears to perform well with new BPs (UCLA data) Etest EA CA me ME/VME Ciprofloxacin Levofloxacin Deak et al JCM :298 All but 1 me were BMD I, Etest R

55 Azithromycin tests Inner zone Outer zone MIC Method Broth microdilution MIC (µg/ml) Etest inner zone 8 Etest outer zone 1 8 Azm Can observe double zones on Etest and DD with some media read inner zone

56 Challenges with testing stool pathogens Azithromycin! Common treatment option, but off-label for Shigella, Salmonella No clinical breakpoints, just ECVs how to report?? Fluoroquinolones! Changing breakpoints for Salmonella spp., need to do off-line testing (test down to 0.06 ug/ml) Recent CDC report: Shigella should have same BP as Salmonella what to do?

57 Azithromycin Breakpoints - WHO, CDC good treatment option for Salmonella, Shigella - Azithromycin concentrates in PMNs, where Salmonella and Shigella located - Successfully used for years, very few clinical failures - CLSI has azithromycin BP for S. Typhi ALONE; ECV for Shigella sonnei and S. flexneri Antimicrobial Agent Disk Content (µg) DD (mm) MIC (µg/ml) S I R S I R S. Typhi Breakpoint S. sonnei ECV S. flexneri ECV Comments

58 Bottom line I get it, it s not easy Some public health responsibility A lot of patient responsibility even if you can t/don t do it in house, send to ref lab. Attempt to culture the bug

59 Culture results

60

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