(AST) Update by Janet Hindler, MCLS MT(ASCP) - SWACM

Antimicrobial SusceptibilityTesting (AST) UpdateJanet Hindler, MCLS MT(ASCP)Sr. Specialist, Clinical MicrobiologyUCLA Medical Centerjhindler@ucla.edu…also consultant with the Associationof Public Health Laboratories

At the conclusion of this talk, youwill be able to……♦Discuss detecting and reporting ofmultidrug-resistant resistant (MDR) gram-negativebacteria.♦Describe current recommendations forreporting vancomycin for Staphylococcusaureus.♦Discuss strategies for coping withcontinual changes in recommendationsfor AST and reporting.

Features…ESBLsMDRA. baumanniiP. aeruginosaE. aerogenesFeatures…MDR KlebsiellaOther MDR GNR

International effort ongoing tostandardize definitions for:♦MDR – multidrug-R(e.g., “R” to ≥ 3 drug classes)♦XDR – extensively drug-R(e.g., “R” to almost all classes but retains “S” to atleast one drug class)♦PDR – pandrug-R(e.g., “R” to all drug classes)Definitions apply to “acquired” (vs. “intrinsic”)) resistanceand to drugs that might be used to treat an infectioncaused by the species.

“Individual facilities should seek appropriateguidance and adopt effective measures that fittheir circumstances and needs.” (page 31)http://www.cdc.gov/ncidod/dhqp/

Specimen: FluidDiagnosis: Surgical wound infxnSerratia marcescensamikacinampicillincefazolinceftriaxoneciprofloxacingentamicinimipenempiper-tazobactamtobramycintrimeth-sulfaMIC (µg/ml)(1 S>32 R>32 R≤0.5 S≤0.25 S≤0.5 S>16 R≤8 8 S1 S≤1/19 SWhat about imipenem? cephalosporins?

Results verified with standard disk diffusion test…CeftriaxCeftazMeroImip

Appendix G. Screening and Confirmatory Testsfor Carbapenemases in EnterobacteriaceaeMHTCLSIM100-S19.

What are the mechanisms of carbapenemresistance in Enterobacteriaceae…♦Carbapenemase (β-lactamase that hydrolyzescarbapenems)– Exmp. KPC (Klebsiella(pneumoniaecarbapenemase)♦Cephalosporinase combined with porin loss– Some cephalosporinases (e.g., AmpC β-lactamases or ESBLs) have low-levellevelcarbapenemase activity– Porin loss limits entry of carbapenem into the cell

Modified Hodge TestS. marcescensweak positiveKPC pos. K. pneumoniaeATCC BAA-1705KPC neg. K. pneumoniaeATCC BAA-1706

Molecular testing revealed SME-2. Where doesSME fit among carbapenemases?MolecularClassABDCarbapenemaseKPCSMEalso IMI, NMC, GESMetallo beta-lactamases(IMP, VIM, GIM, SPM)OXAFound in:K. pneumoniae andother EnterobacteriaceaeS. marcescensEnterobacteriaceaeP. aeruginosa,Enterobacteriaceae,Acinetobacter,S. maltophiliaAcinetobacter baumanniiAdapted from Queenan & Bush. 2007. Clin Microbiol Rev. 20:440.

Some features of carbapenemases…CarbapenemaseKPCSMEMBLHydrolysis profile3 rd , 4 thgen cefs++/-+Aztreonam++-Inhibition profileEDTA--+Clavulanicacid++-3 rd gen cefs, , ceftriaxone, cefotaxime, ceftazidime4 th gen cef, , cefepimeMBL, metallo beta-lactamaseAdapted from Queenan & Bush. 2007. Clin Microbiol Rev. 20:440.

What is unique about SME as comparedto KPC?♦ Gene located on chromosome (vs. KPC on plasmid)♦ Less hydrolysis of ceftriaxone, cefotaxime,ceftazidime, cefepime than KPC (S.(marcescens “S”to these in vitro; ? activity in vivo)Majiduddin et al. 2005. Antimicrob Agents Chemother. 59:3421.Queenan et al. 1992. Antimicrob Agents Chemother. 44:3035.♦ Infrequently encountered:– 2000-20052005– 50,881 Enterobacteriaceae isolated from N. America, LatinAmerica, Europe– 5 SME (S.(marcescens)!Castanheira et al. 2008. Antimicrob Agents Chemother. 52:570.

Specimen: FluidDiagnosis: Surgical wound infxnSerratia marcescensFinal reportamikacinampicillincefazolinceftriaxoneciprofloxacingentamicinimipenempiper-tazobactamtobramycintrimeth-sulfaMIC (µg/ml)(1 S>32 R>32 R≤0.5 S≤0.25 S≤0.5 S>16 R≤8 8 S1 S≤1/19 SReport comment:“Imipenem-R R is due tocarbapanemase production(but not KPC). Theeffectiveness of β-lactams intreating infections due tocarbapenemase-producingproducingS. marcescens has not beenestablished. ID consultsuggested.”

Selective Reporting OptionSerratia marcescensamikacinampicillincefazolinceftriaxoneciprofloxacingentamicinimipenempiper-tazobactamtobramycintrimeth-sulfaMIC (µg/ml)(1 S>32 R>32 R≤0.5 S≤0.25 S≤0.5 S>16 R≤8 8 S1 S≤1/19 SSome may suppresscarbapenem result if “S” toceftriaxone and piperacillin-tazobactam….but, always report“unexpected resistance”.(CLSI M100-S19. Table 1.Note 1. p 33. )

http://www.cdc.gov/ncidod/dhqp/ar_kp_lab.html

MMWR March 20, 2009 (1)http://www.cdc.gov/mmwr/CRE = carbapenem-resistantresistantEnterobacteriaceae

MMWR March 20, 2009 (2)LaboratoryResponsibilities…Use reliable testmethods and reportCRE promptly!Surveillance

Specimen: BloodDiagnosis: Neutropenic feverKlebsiella pneumoniaeamikacinampicillincefazolinceftazidimeceftriaxoneciprofloxacingentamicinimipenempiper-tazobactamtobramycintrimeth-sulfaMIC (µg/ml)(32 R>32 R>32 R>32 R>32 R>2 R2 S>16 R128 R>10 R>4/76 RWhat about aminoglycosides?

http://www.clsi.org/Content/NavigationMenu/Committees/Microbiologygy/AST/AST.htm

http://www.bsac.org.uk/susceptibility_testing/guide_to_antimicrobial_susceptibility_testing.cfm

AAC(6’) “common” in S. marcescensDavid Livermore (BSAC website)

Specimen: BloodDiagnosis: Neutropenic feverKlebsiella pneumoniaeFinal Reportamikacinampicillincefazolinceftazidimeceftriaxoneciprofloxacincolistingentamicinimipenempiper-tazobactamtigecyclinetobramycintrimeth-sulfaMIC (µg/ml)(32 R>32 R>32 R>32 R>32 R>2 R≤0.12*2 S>16 R128 R1 S>10 R>4/76 RReport comment:“*No interpretive criteria availableImipenem resistance NOT due tocarbapenemase production.The effectiveness of gentamicin intreating infections due to K.pneumoniae that are gentamicin-S,amikacin-R R and tobramycin-R R hasnot been established. ID consultsuggested.”

Specimen: Tracheal AspirateDiagnosis: PneumoniaAcinetobacter baumanniiMIC (µg/ml)(amikacin>32 Rceftriaxone>32 Rcolistin≤0.5 Sciprofloxacin>4 Rgentamicin>16 Rimipenem>16 Rpiper-tazobactam>512 Rtigecycline 2*tobramycin>16 Rtrimeth-sulfa≤4/76 RWhat about tigecycline?* no interpretive criteria

Tigecycline – FDA Breakpoints(no CLSI breakpoints)Tigecyclinehttp://www.wyeth.com(Prescribing Information)

What should we know about testing/ reportingtigecycline on Acinetobacter baumannii?♦ Currently, no FDA approval for use in treatingAcinetobacter infections♦ Only breakpoints for Acinetobacter spp. availableare published by AST standards-type type organizationare from British Society for AntimicrobialChemotherapy (BSAC)http://www.bsac.org.uk/_db/_documents/– MIC (µg/ml)(≤1 1 S, 2 I, >2 2 R– Peleg et al. recommends using these for A. baumanniiinfections other than bloodPeleg et al. 2008. Clin Microbiol Rev. 21:538.

Why shouldn’t t we use the BSAC Acinetobacterspp. breakpoints for blood isolates?♦Maximum blood concentration =0.63 µg/ml– Prudent NOT to report blood isolates withMICs >0.5 µg/ml as “S”– Perform MIC not disk diffusion for bloodisolatesPeleg et al. 2008. Clin Microbiol Rev. 21:538.

What are the activities tigecycline andtetracyclines against MDR A. baumannii?%Susceptible (N=93)Tigecycl*MinoDoxyA-SAmikImipCiproPoly B9583562038200100*Used MIC breakpoint (S(≤2 µg/ml) for Enterobacteriaceae fromtigecycline product package insert (Wyeth Pharmaceuticals Inc.)Scheetz et al. 2007. Antimicrob Agents Chemother. 51:1621.

What are the activities of tigecycline andminocycline against MDR A. baumannii?Tested:Amp-sulbPip-tazoAztreCeftriaxCeftazCefotaxCefepimeImipMeroCiproOfloxSXTTetraGentTobAmikIsolatePDRXDRIPM-STotalN464064150TigecyclineMIC (µg/ml)(Range0.5-80.25-40.03-20.03-890%2222Range0.25-160.25-32≤0.06-16≤0.06-32MinocyclineMIC (µg/ml)(Excluding tigecycline and minocycline….PDR = R to all including colistinXDR = only S to colistinIPM-S S = variable90%1616816%S8.73060.936.6Arroyo et al. 2009. Antimicrob Agents Chemother. 53:1295.

Method-related discordance in tigecyclineMICs for Acinetobacter baumannii (N=227);Etest vs. broth microdilution (BMD)MIC 90 : Etest = 4 µg/ml BMD = 1 µg/mlPillar et al. 2008. J Clin Microbiol. 46:2862.See also...Casalet al. 2009. J Antimicrob Chemother. 64:69.

Specimen: Tracheal AspirateDiagnosis: PneumoniaAcinetobacter baumanniiFinal ReportMIC (µg/ml)(amikacin>32 Rceftriaxone>32 Rcolistin≤0.5 Sciprofloxacin>4 Rgentamicin>16 Rimipenem>16 Rpiper-tazobactam>512 Rtigecycline 2*tobramycin>16 Rtrimeth-sulfa≤4/76 RReport comment:“*No interpretive criteria;Infectious Diseasesconsult suggested.”

Specimen: Pleural fluidDiagnosis: PneumoniaStaphylococcus aureusMIC (µg/ml)(clindamycin>8 Rerythromycin>8 Roxacillin>16 RpenicillinRvancomycin 2 SShould we do anything aboutvancomycin MIC of 2 µg/ml?

S. aureus - VancomycinMIC Interpretive Criteria (µg/ml)(Susceptible≤2VSSA≤1 vs 2 µg/mlhVISAIntermediate4-8VISAResistant≥16VRSACLSI M100-S19; Table 2C.hVISA (h=heteroresistant) no CLSI recommendations for detecting hVISA

Why does it matter if vancomycinMIC is 1 vs. 2 µg/ml?♦ Based on PK/PD, , there may be different clinicaloutcomes if vancomycin used for therapy♦ Infectious Diseases Society of America (IDSA)Guidelines; if vancomycin MIC is:– ≤1 µg/ml– maintain vancomycin trough serumconcentration of 15-20 mg/L– ≥2 µg/ml- use alternative treatmentRybak et al. Clin Infect Dis. 2009. 49:325♦ Increasing numbers of isolates with susceptibleMICs of 1 and 2 µg/ml (as compared to ≤0.5µg/ml)- referred to as vancomycin “creep”♦ Some suggest lowering the breakpoint to ≤1 µg/mlfor “S”.

Vancomycin MICs are “Creeping up”MRSA Blood Isolates 2001-2005 2005 (N=662)MIC testing performed by Etest methodSteinkraus et al. 2007. J Antimicrob Chemother. 60:788.See also…Wang et al. 2006. J Clin Microbiol. 44:3883.

Outcomes of Vancomycin Therapy in92 Patients with MRSA Bacteremia(2005-2007)2007)OutcomeOverall failureHospital length of stayVAN MIC ≥1.5(66 patients)24 (36.4)*21 (9.0-43.0)VAN MIC

What methods should we use for routinetesting of vancomycin and S. aureus?♦ Is Etest best?– More recent outcome databased on Etest♦ Method variability– Etest MICs higher than CLSIreference method MICs♦ Reproducibility of MIC testsgenerally +/- 1 two-fold dilutionMIC = 1.5 µg/ml

Vancomycin MIC (N=101 MRSA)Etest MICs > Reference Broth Microdilution and Agar Dilution MICsPrakash et al. 2008. Antimicrob Agents Chemother. 52:4528.See also…Hsu et al. 2008. Intl J Antimicrob Agents. 32:378.

S. aureus - Vancomycin MIC DistributionUCLA 2008 (889 MSSA, 662 MRSA)90% of isolates80706050403057.448.742.550.5MSSAMRSA201000.1 0.8=4Vancomycin MIC (ug/ml)CLSI reference broth microdilution method

Specimen: Pleural fluidDiagnosis: PneumoniaStaphylococcus aureusMIC (µg/ml)(clindamycin >8 Rdaptomycin0.5 Serythromycin>8 Rlinezolid1 Soxacillin>16 RpenicillinRvancomycin 2 S*Final report“*Vancomycin MIC determined by Etest method”

Specimen: BloodDiagnosis: EndocarditisStaphylococcus aureusMIC (µg/ml)(clindamycin>8 Rerythromycin>8 Roxacillin>16 RpenicillinRvancomycin ≤0.5 SCould this be hVISA?

What is hVISA*?♦S. aureus that show….– vancomycin MIC ≤2 µg/ml (S) by conventionalMIC tests– subpopulation of cells with MICs 4-8 µg/ml– associated with increase production of biofilm;more tolerant to “killing”;; poor patientoutcomes*heteroresistant VISA

What might an hVISAinoculum look like?Too few “NS” cells to detect withstandard inoculum size in routineAST (MIC & DD results will be “S”);often slower growingSIR≤0.5124816≥32= 1 bacteriumVancomycin MIC (µg/ml)(NS, not susceptible

What methods have been used todetect hVISA?♦ Population analysis/AUC (PAP/AUC)– Place high inoculum on agar with varying vancomycinconcentrations and divide by area under the populationcurve (gold standard)♦ Etest (test vancomycin and teicoplanin)– Macro method - high inoculum (McF(#2) on BHI– GRD (glycopeptide resistance detection)See Yusof et al. 2008. JCM. 46:3042♦ Screen plates– MHA + 5 µg/ml teicoplanin– Other screen platesGRD Etest

Outcomes in 250 patients withBacteremia due to hVISA or VS-MRSAInfection-attributable deathHospitalization duration, daysBacteremia, dayshVISA(N=27)12 (44)*12 (0-207)12 (1-123)123)VS-MRSA(N=223)81 (36)12.5 (0-184)2 (1-92)p.4.8.005Endocarditis5 (19)8 (4).007Osteomyelitis7 (26)16 (7).006*No. (%) of patients or median value (range)hVISA identified with Etest macromethodMaor et al. 2009. J Infect Dis. 199:619.

Pt. X – 4 MRSA Blood Isolates (same PFGE)Endocarditis - failed vancomycin and daptomycin RxIsolateDateDaptoBMDfreshMIC (µg/ml)(VancBMDfreshVancEtest*Etest*freshfrozenhVISA EtestMacroGRDPAP9/16≤0.5 S1 S2 S2 S---11/5≤0.5 S2 S2 S2 ShVISA-hVISA12/44 NS2 S4 I2 ShVISAhVISAhVISA12/64 NS4 I8 I3 (I)hVISAhVISAhVISABMD, broth microdilution* Std. method, McFarland 0.5 / MHAFresh, isolate tested when recoveredFrozen, isolate tested after freezingPAP, population analysisTenover et al. 2009.Intl J Antimicrob Agents. 33:564.

CLSI Retesting Rule♦ForS. aureus, , vancomycin ”S”isolates may become vancomycin ”I”during prolonged therapy.Suggestion:Test subsequent isolates of S. aureus fromsimilar body site after 3-434 days to see ifisolate is still vancomycin-S.CLSI M100-S19. Introduction.

Specimen: BloodDiagnosis: EndocarditisStaphylococcus aureusMIC (µg/ml)(clindamycin>8 Rerythromycin>8 Roxacillin>16 RpenicillinRvancomycin ≤0.5 SFinal report“hVISA identified by Macro Etest method; MacroEtest performed at Dr. Smith’s s request”

How can we handle all of this????“Need…..Artificial Intelligence”!♦ Flag atypical / inconsistent results♦ Suggest confirmatory tests♦ Report appropriate drugs♦ Edit “S” or “I” results to “R”♦ Add comments to reportand♦ Informed Clinical Laboratory Scientists!AST

Summary (1)♦ Carbapenem resistance in Enterobacteriaceae maybe due to carbapenemases or combinations of otherβ-lactamases and porin changes.♦ SME carbapenemases are uncommon, do notreadily hyrdolyze 3 rd and 4 th generationcephalosporins and can be modified Hodge testpositive.♦ The effectiveness of gentamicin in gentamicin-S,amikacin-R R and tobramycin-R R Enterobacteriaceae isnot known.♦ There are currently no CLSI or FDA breakpoints forinterpreting results for tigecycline withAcinetobacter spp.

Summary (2)♦ Serious infections caused by MRSA withvancomycin MICs of >1 µg/mlmay not respond aswell to vancomycin therapy as MRSA withvancomycin MICs of ≤1 µg/ml.♦ Different methods may result in differentvancomycin MICs for vancomycin-S S. aureus.Etest produces higher vancomycin MICs thanCLSI broth microdilution reference method.♦ There are currently no CLSI or other standardrecommendations for testing for hVISA.

"...there is nothing so satisfying to thespirit, so defining of our character thangiving our all to a difficult task!President Barack Obama, , 1/20/09