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Susceptibility Testing of Mycobacteria,Susceptibility Testing of Mycobacteria,NocardiaeNocardiae, and Other Aerobic , and Other Aerobic
ActinomycetesActinomycetesyyCLSI M24CLSI M24--A2, March 2011A2, March 2011
(largely taken from a presentation prepared by Gail L. Woods, MD
and Barbara Brown-Elliott, MS, MT(ASCP)SM)
Edward Desmond, CDPH
1
June 2011
M24M24--A2 AuthorsA2 AuthorsM24M24--A2 AuthorsA2 Authors
• Gail L. Woods• Barbara Brown-Elliott
• Gaby E. Pfyffer• John C. Ridderhof
• Patti Conville• Edward P. Desmond
G ldi S H ll
• Salman H. Siddiqi• Richard J. Wallace• Nancy G Warren• Geraldine S. Hall
• Grace Lin• Nancy G. Warren• Frank G. Witebsky
Special thanks to Tracy Dooley, CLSI Senior Standards Administrator and Staff Liason for Microbiology
2
At the conclusion of this At the conclusion of this talk, you will be able to…..talk, you will be able to…..
• Discuss the indications for susceptibility• Discuss the indications for susceptibility testing of MTBC, NTM, & aerobic actinomycetesactinomycetes
• Discuss the methods for susceptibility testing of MTBC NTM & aerobictesting of MTBC, NTM, & aerobic actinomycetes
• Accurately interpret and report results• Accurately interpret and report results• Establish and implement a QC program
3
Program OutlineProgram Outlinegg
• Mycobacterium tuberculosis complexy p• Nontuberculous mycobacteria• Nocardia & other aerobic• Nocardia & other aerobic
actinomycetes
4
Susceptibility Testing for MTBCSusceptibility Testing for MTBCp y gp y g
• Standard based on proportion methods• Standard based on proportion methods– Agar (INH, RMP, EMB) or radiometric (PZA)
• Resistance:– “decrease in susceptibility of sufficient degree to be
reasonably certain that the strain concerned is different from a sample of wild strains of human type that have never come into contact with the drug.”never come into contact with the drug.
– >1% of an inoculum of bacterial cells in the presence of a “critical concentration” of anti-TB drug
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Critical ConcentrCritical Concentration
• Adopted by international convention• Adopted by international convention
• Lowest concentration of anti-TB drugs that inhibit 95% of “wild strains” of MTBC that have never been exposed to the drugs, but not inhibiting strains isolated from patients failing to respond to therapyisolated from patients failing to respond to therapy
• Test susceptibility of MTBC to the critical concentration of drug specific for the test method you are using
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Susceptibility Testing of MTBCSusceptibility Testing of MTBCUniq e AspectsUniq e AspectsUnique AspectsUnique Aspects
• Testing is performed at 1 (or 2) concentrations• Testing is performed at 1 (or 2) concentrations• “Critical” concentrations established years ago, &
values may differ depending on test mediumy p g• No uniform consensus regarding clinical
relevance of testing other concentrations• Agar proportion uses %age calculation to
determine R or S• Radiometric method for PZA testing uses drug• Radiometric method for PZA testing uses drug-
specific calculation procedure to determine R or S
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Agar Proportion MethodAgar Proportion MethodLimitationLimitationLimitationLimitation
• Not rapid• Not rapid• Broth method with shorter incubation time is
recommended standard of practice inrecommended standard of practice in industrialized countries
• CDC goal: report results of primary drugs• CDC goal: report results of primary drugs within 15-30 days of receipt of specimen
7 14 days after isolation of MTBC (ideal)– 7-14 days after isolation of MTBC (ideal)
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Agar Proportion MethodAgar Proportion MethodCurrent UsesCurrent Uses
• Confirm commercial broth system result if necessary• Confirm commercial broth system result, if necessary
• Test drugs &/or concentrations of drugs not available in commercial systems
• Standard against which new methods are evaluated &• Standard against which new methods are evaluated & for characterizing in vitro susceptibility of “new” anti-TB drugs
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MTBC Susceptibility TestingMTBC Susceptibility TestingRecommended Dr gsRecommended Dr gsRecommended DrugsRecommended Drugs
• PREVIOUS:• PREVIOUS: • 2 concentrations of INH (critical & higher); • Might consider INH, RMP, EMB onlyg , , y
• NEW: Initially test all primary drugs at critical concentration– INH, RMP, EMB, PZA– PZA resistance more common, not predictable,
always requires modification of treatment
10
Primary Drugs for FDAPrimary Drugs for FDA--Cleared Cleared Commercial Broth SyCommercial Broth Systems
DrugSystem and Concentration (µg/ml)
BACTEC BACTEC V TREK
460TB MGIT 960VersaTREK
INH 0.1 0.1 0.1
RMP 2.0 1.0 1.0
EMB 2.5 5.0 5.0
PZA 100 100 300
11
Testing Frequency for Primary DrugsTesting Frequency for Primary DrugsTesting Frequency for Primary Drugs Testing Frequency for Primary Drugs
• Initial isolate from every patient
• Repeat if cultures do not convert to negative after 3 months of therapy or earlier if there is clinical evidence of failure
• Confirm if initially resistant, by repeat testing (?) or other means, e.g. molecular
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Initial Results Indicate Initial Results Indicate Resistance to ≥1 DrugResistance to ≥1 DrugResistance to ≥1 DrugResistance to ≥1 Drug
• Exclude bacterial contaminant and NTM– Examine vial/tube: MTBC usually grows in
clumps & broth tends to remain clear– Prepare AFB-stained smear: cording suggests
MTBC di d di t ib ti d lMTBC; dispersed distribution or random, loose clumps throughout smear suggests NTMS b lt– Subculture
13
Initial Results Indicate Initial Results Indicate Resistance to ≥1 DrugResistance to ≥1 DrugResistance to ≥1 DrugResistance to ≥1 Drug
• Determine need for repeat testing– Is molecular testing available?—beacons, line
probe or sequencing?• If repeat or molecular testing planned:
– Report initial resistance, indicating results preliminary & confirmation in progress
– Testing secondary agents simultaneously strongly recommended
14
SecondSecond--line Drug Testingline Drug Testingg gg g
• Indications– Resistance to RMP or any 2 primary agentsy p y g
– NEW: Do second-line testing when there is gmono-resistance to critical concentration of INH if therapy with fluoroquinolone planned
15
SecondSecond--line Drug Testingline Drug TestingRR Ri h CRi h C t iResourceResource--Rich CounRich Countries
T t ≥1 d f h l l hi h• Test ≥1 drug from each class plus higher concentrations of INH & EMB– Exception is cycloserine: testing not recommended due
to technical issues
– “Each class”: e.g. fluoroquinolones, injectables• Avoid “piecemeal” approach
If t d i h i di t l d t l b ith• If not done in-house, immediately send to lab with second-line drug testing expertise
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SecondSecond--line Drugs Listed in M24*line Drugs Listed in M24*gg
• Capreomycin: 10 0/10 0 (7H10 conc/7H11 conc)Capreomycin: 10.0/10.0 (7H10 conc/7H11 conc)• Ethionamide: 5.0/10.0• Ethambutol: 10.0/10.0 (higher concentration than primary)
K i 5 0/6 0 ( l t ti f ik i )• Kanamycin: 5.0/6.0 (class representative for amikacin)• Ofloxacin: 2.0/2.0 (class representative for
fluoroquinolones)• PAS: 2.0/8.0• Rifabutin: 0.5/0.5
– Some test 1-2 µg/ml; however clinical significance is unknown– Some test 1-2 µg/ml; however, clinical significance is unknown• Streptomycin: 2.0 & 10.0/2.0 & 10.0
*Concentrations (µg/ml) in 7H10 agar/7H11 agar
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SecondSecond--line Drug Testingline Drug TestingNe in M24Ne in M24 A2A2New in M24New in M24--A2A2
• Amikacin added (4 µg/ml in 7H10;• Amikacin added (4 µg/ml in 7H10; agar/breakpoint in 7H11 not established)
• Resistance to kanamycin may not indicate resistance to amikacinresistance to amikacin– Consider testing both aminoglycosides
18
SecondSecond--line Drug Testingline Drug TestingNe in M24Ne in M24 A2A2New in M24New in M24--A2A2
Fluoroquinolone 7H10 (µg/ml) 7H11 (µg/ml)Fluoroquinolone 7H10 (µg/ml) 7H11 (µg/ml)
Levofloxacin 1.0 ND
Moxifloxacin 0.5 0.5
Ofloxacin 2.0 2.0
1. Fluoroquinolone tested should be selected based on consultation with physician treating most resistant TB
2 Testing levofloxacin at 1 0 µg/ml = testing ofloxacin at 2 0 µg/ml2. Testing levofloxacin at 1.0 µg/ml testing ofloxacin at 2.0 µg/ml3. # studies with moxifloxacin by agar proportion are limited; more studies needed
ND=not determined
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SecondSecond--Line Drug Testing in Line Drug Testing in Commercial Broth SystemsCommercial Broth SystemsCommercial Broth SystemsCommercial Broth Systems
• Amikacin: 1.0/1.0• Capreomycin: 1.25/2.5
Ethi id 2 5/5 0
• Linezolid: 1.0/1.0• Moxifloxacin: 0.5/0.25
Ofl i 2 0/2 0• Ethionamide: 2.5/5.0• Kanamycin: 5.0/2.5• Levofloxacin: 2 0/1 5
• Ofloxacin: 2.0/2.0• Rifabutin: 0.5/0.5
• Levofloxacin: 2.0/1.5
Appendix A
Concentrations (µg/ml) are interpretive criteria, based on multicenter
studies, for BACTEC 460/MGIT 960 (neither is FDA-cleared for testing second-line drugs)
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for testing second-line drugs)
M24M24--A2 Appendix A*A2 Appendix A*Fl i l T tiFl i l T tiFluoroquinolone TestingFluoroquinolone Testing
• Ofloxacin is the class representative• Ofloxacin is the class representative• Test at least 1 of the 3 (consult specialist in managing
drug-R TB)• Moxifloxacin S at 0.25 µg/ml by MGIT 960 =
levofloxacin S• If moxifloxacin R at 0 25 µg/ml by MGIT 960 consider• If moxifloxacin R at 0.25 µg/ml by MGIT 960, consider
testing at 0.5, 1, 2, and 4 µg/ml to determine level of resistance (studies needed to assess clinical efficacy of moxifloxacin for isolates with MIC of 0.5-4 µg/ml)
*Footnote
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SecondSecond--line Drug Testingline Drug TestingReso rceReso rce Limited Co ntries*Limited Co ntries*ResourceResource--Limited Countries*Limited Countries*
• Prioritize choices of drugs to testg
• 1st : INH, RMP
• 2nd : EMB, PZA, streptomycin
• 3rd : amikacin, kanamycin, capreomycin, fluoroquinolone (based on surveillance data)fluoroquinolone (based on surveillance data)
*Appendix B
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Agar Proportion MethodAgar Proportion MethodCh i M24Ch i M24 A2A2Changes in M24Changes in M24--A2A2
• Select & prepare stock solution of drugs to test– Details in Table 2; example calculation in Appendix C
• Agar medium: 7H10 or 7H11• Agar medium: 7H10 or 7H11– Preparation & plating moved to Appendix D– Drug-containing disks moved to Appendix E– Liquid drug moved to Appendix F
• Inoculum density for indirect method = 0.5 to 1.0McFarland standardMcFarland standard
• Incubation temperature 35-37C
23
Susceptibility Testing of MTBCSusceptibility Testing of MTBCRes lt Reporting What’s Ne ?Res lt Reporting What’s Ne ?Result Reporting: What’s New?Result Reporting: What’s New?
Shortened suggested comments for INH RShortened suggested comments for INH-R, depending on # of concentrations tested
– If test result indicates R at critical concentration to INH,If test result indicates R at critical concentration to INH, and high conc not tested: “This test result indicates the presence of at least low level resistance to INH.”If t t lt i di t hi h l l R t INH “Th t t– If test results indicate high-level R to INH: “These test results indicate high-level resistance to INH.”
– For both: “A specialist in the treatment of drug-R TBFor both: A specialist in the treatment of drug R TB should be consulted concerning the appropriate therapeutic regimen and dosages.”
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Susceptibility Testing of MTBCSusceptibility Testing of MTBCQ alit ControlQ alit ControlQuality ControlQuality Control
Al a s test pan s sceptible isolate• Always test pan-susceptible isolate– H37Rv (ATCC 27294)
H37Ra: avirulent unique HPLC pattern– H37Ra: avirulent, unique HPLC pattern• Consider ATCC BAA-812 if testing INH at 2
concentrations– R to critical concentration/S to higher concentration
• Avoid working with strains resistant to >2 drugs or exhibiting high-level resistance to single drug
25
Susceptibility Testing of MTBCSusceptibility Testing of MTBCQ alit ControlQ alit ControlQuality ControlQuality Control
Storage of controls prepare s spension in• Storage of controls: prepare suspension in suitable stabilizer, distributed in small aliquots in multiple vials & maintained at -20C or belowmultiple vials, & maintained at 20C or below (never in self-defrosting freezer)
• Test frequency– Pan-S (H37Rv): once each week patient isolates tested– R (BAA-812): can be less frequent (eg monthly) unless
problem identified
26
Molecular Detection of Drug Molecular Detection of Drug Resistance in MTBCResistance in MTBCResistance in MTBCResistance in MTBC
• Commercially available methods– Real-time PCR, PCR/line probe– Not yet FDA clearedNot yet FDA cleared
• Test positive cultures or AFB smear-positive sputum• Identify MTBC and detect mutations associated with INH &
RMP resistanceRMP resistance• Interpret negative mutation results with caution: resistance
may be caused by different mutations• Detection of MTBC DNA does not necessarily = viability• May perform on mixed/contaminated cultures
27
Molecular Detection of DrugMolecular Detection of Drug--R R MTBC: Possible IndicationsMTBC: Possible IndicationsMTBC: Possible IndicationsMTBC: Possible Indications
• Patients with wide range of contacts who may have spread infection to many others
• Patients suspected of having drug-R diseaseHistory of previous treatment– History of previous treatment
– From countries or ethnic groups with increased drug-R– Not responding well to treatmentNot responding well to treatment– Exposed to MDR-TB
28
Nontuberculous Nontuberculous MycobacteriaMycobacteria
29
Antimycobacterial Susceptibility Testing Antimycobacterial Susceptibility Testing (AST) of Nontuberculous Mycobacteria (AST) of Nontuberculous Mycobacteria ( ) y( ) y
(NTM)(NTM)
Rapidly Growing Mycobacteria (RGM) ~ 70 species 70 species
Species grow < 7 days Slowly Growing Mycobacteria (SGM)Slowly Growing Mycobacteria (SGM)
~ 69 speciesSpecies grow > 7 daysSpecies grow > 7 days
More than one half identified since 1990
30
More than one half identified since 1990
Rapidly Growing MycobacteriaRapidly Growing MycobacteriaIdentification to species or group Identification to species or group p g pp g pimportant to determine treatmentimportant to determine treatment
Species grow < 7 daysM. fortuitum groupg pM. chelonae/abscessus groupM smegmatis groupM. smegmatis groupM. mucogenicum groupPigmented RGM (~35 species asPigmented RGM (~35 species as of 2011)
31
Rapidly Growing Rapidly Growing MycobacteriaMycobacteriaMycobacteriaMycobacteria
• Non-pigmented Pathogens
M. fortuitum groupM f t itM. fortuitumM. peregrinumM senegalense/conceptionense*M. senegalense/conceptionenseM. setense
* Same genetically Same genetically_____________________________Wallace, et. al., J. Clin. Microbiol. 2005
32
Rapidly Growing MycobacteriaRapidly Growing Mycobacteria
M. fortuitum group (formerly Third Biovariant Group)M i M b i bM. porcinum M. brisbanense M. houstonense M. neworleansenseM b i k i M tiM. boenickei M. septicumM. mageritense*
*Phylogenetically distinct; may be included withM li k iM. wolinskyi___________________________Schinsky, et al., IJSEM, 2004
33
Rapidly Growing Rapidly Growing MycobacteriaMycobacteriaMycobacteriaMycobacteria
• Nonpigmented Pathogens
M. chelonae / abscessus group
M. chelonae M. abscessus subsp. abscessusM. immunogenum M. abscessus subsp. bolletiiM salmoniphilum (formerly M massilienseM. salmoniphilum (formerly M. massiliense,
M. bolletii)_______________________________________Adekambi, et al., J. Clin. Microbiol. 2003 Brown-Elliott, et al., CMR, 2002de a b , et a , J C c ob o 003 o ott, et a , C , 00Adekambi, et al., J. Clin. Microbiol. 2004 Whipps, et al., IJSEM, 2007Adekambi, et al., IJSEM 2006 Leao, et al., J. Clin. Microbiol 2009Leao, et al., IJSEM 2011
34
Rapidly Growing Rapidly Growing M b t iM b t iMycobacteriaMycobacteria
M mucogenicum groupM. mucogenicum group (formerly M. chelonae-like Organism)
M. mucogenicumM. aubagnenseM h iM. phocaicum
________________________________Adekambi et al IJSEM 2006Adekambi, et al., IJSEM, 2006Springer, et al., J. Clin. Microbiol., 1995
35
Rapidly Growing Rapidly Growing MycobacteriaMycobacteriaMycobacteriaMycobacteria
Late Pigmenting Species: M. smegmatis groupg g p g g p
M. smegmatis (formerly M. smegmatissensu stricto)sensu stricto)
M. goodiiM. wolinskyi**
** Non-pigmenting phylogenetically distinct (may be included with M. mageritense)
_________________________________Brown BA, et al., IJSB, 1999
36
, , ,
Rapidly Growing Rapidly Growing M b t iM b t iMycobacteriaMycobacteria
Early Pigmenting Speciesy g g p(~35 species as of 2010)
M. aurum/neoaurum* M. canariasense*M. bacteremicum* M. cosmeticum*M flavescens M monacense*M. flavescens M. monacenseM. vaccae “M. lacticola”*M. phlei M. psychrotoleransM th i tiblM. thermoresistible
* Proven pathogens
37
___________________________________Tortoli, E., FEMS Immunol. Med Microbiol. 2006Brown-Elliott et al., J. Clin. Microbiol. 2010
Recommendation for Which Recommendation for Which Isolates to TestIsolates to Test
Follow ATS criteria for respiratory samples
Isolates to TestIsolates to Testp y p
Generally multiple (+) samples 2 (+) sputa or 1 Bronch( ) p (+) Transbronch / lung bx Single (+) sputum not likely to be significant
Clinically significant isolates from blood, sterile body fluids, skin and soft tissueRepeat susceptibility after 6 months if (+)
cultures continue_____________________
38
Griffith, et al., ATS/IDSA Statement 2007
Susceptibility Testing of RGMSusceptibility Testing of RGM
Broth Microdilution is recommended “Gold Standard”
Match McFarland 0 5 turbidity standardMatch McFarland 0.5 turbidity standardMay require use of beads to homogenize
2 f ld dil ti i CAMHB 2 fold dilutions in CAMHB Organism concentration 105 CFU/mL or 104
CFU/well in 0.1 mL volume Incubation 28-30o C / 3 days/room air_____
39
y _____CLSI, M24-A2, 2011
Antimicrobial Susceptibility Antimicrobial Susceptibility Testing of RGMTesting of RGMTesting of RGM Testing of RGM (cont’d)
CLSI M24 A2, 2011
Clarithromycin MICs 3 days (mutational resistance) =3 days, (mutational resistance)
Point mutation adenine 2058 or 2059 in 23S rRNA geneFi l di t 14 d Final reading at 14 days
Inducible clarithromycin resistance(erm gene) at 14 days(erm gene) at 14 days
Especially M. abscessus__________________________________________________________
Wallace et al Antimicrob Agents Chemother 1996
40
Wallace et al., Antimicrob Agents Chemother, 1996.Nash, et al., Antimicrob Agents Chemother, 2009.Nash et al., Antimicrob Agents Chemother., 2006.CLSI M24-A2, 2011.
Clarithromycin ResistanceClarithromycin ResistanceM fortuitum / M abscessusM fortuitum / M abscessus rRNA methylase gene
M. fortuitum / M. abscessusM. fortuitum / M. abscessus rRNA methylase geneerm(39) M. fortuitumConfers inducible macrolide
resistance erm (41) M. abscessus No erm gene in M. chelonae No erm gene in M. chelonae____________________________Nash, et al., Antimicrob Agents Chemother. 2009
41
as , et a , t c ob ge ts C e ot e 009
ermerm Gene in Gene in M. abscessusM. abscessus
• Approximately 85% M. abscessus pp y(subsp. abscessus) contain inducible erm geneinducible erm gene
• Isolates of M. abscessus subsp. bolletii (formerly M massiliense andbolletii (formerly M. massiliense andM. bolletii) do not contain inducible erm gene
_______________________________
42
Nash, et al., Antimicrob Agents Chemother, 2009
ermerm Gene in Gene in M. abscessusM. abscessus( t’d)(cont’d)
•Clinical SignificancegPatients with isolates containing ermgene have delayed treatmentgene have delayed treatment response and possible failures compared to those patients whosecompared to those patients whoseisolates DO NOT contain functionalerm gene______________________
Koh, W-J, et al. Am. J. Resp. Crit. Care Med. 2011Leao, SC, et al. IJSEM. 2010
43
Leao, SC, et al. IJSEM. 2010
Clinical Significance of Clinical Significance of ermerm Gene Gene inin M abscessusM abscessusin in M. abscessusM. abscessus
• Treatment response rates in clarithromycin-Treatment response rates in clarithromycincontaining regimens are higher in patients with M. abscessus subsp. bolletii thanpthose with M. abscessus subsp. abscessuslung diseaseg
• Proportion of patients with sputumconversion and negative sputum cultures88% with M. abscessus subsp. bolletii comparedto 25% with M. abscessus subsp. abscessus
44
Clinical Significance of Clinical Significance of ermerm Gene Gene inin M abscessusM abscessusin in M. abscessus M. abscessus (cont’d)
• M abscessus lung disease has beenM. abscessus lung disease has beenconsidered a chronic incurable infection but this may not be true withbut this may not be true with M. abscessus subsp. bolletii.M b b b d• M. abscessus subsp. abscessus and M. abscessus subsp. bolletii associatedwith CF strains_____________________ Koh, W-J, et al. Am. J. Resp. Crit. Care Med. 2011Bastian S et al Antimicrob Agents Chemo 2011
45
Bastian, S, et al. Antimicrob. Agents. Chemo. 2011
Changes to Broth microdilution RGM Changes to Broth microdilution RGM MIC breakpoints (µg/mL)MIC breakpoints (µg/mL)MIC breakpoints (µg/mL)MIC breakpoints (µg/mL)
Antimicrobial Agent Susceptible Intermediate Resistant
Doxycycline/Minocycline < 1 2-4 > 8Imipenem, Meropenem < 4 8-16 > 32TMP-Sulfamethoxazole < 2/38 - > 4/76Tobramycin < 2 4 > 8M ifl i < 1 2 > 4Moxifloxacin < 1 2 > 4___________________________________________________CLSI, M24-A2, 2011
46
, ,
Reporting MICs of RGMReporting MICs of RGM
Imipenem
p gp g
Imipenem– New breakpoint (I = 8-16 µg/mL) to allow
reporting in all species (MICs more p g p (reproducible)
– Report for M. fortuitum group - If MIC > 8 µg/mL - repeat/confirm
Tobramycin– Report only for M. chelonae– If MIC > 4 µg/mL – repeat/confirm_______
47
CLSI, M24-A2, 2011
Reporting MICs of RReporting MICs of RGM
Amikacin with M. abscessus– If MIC > 64 ug/mL – repeat/confirmIf MIC > 64 ug/mL repeat/confirm
ClarithromycinT ili d i t ith M f t it– Trailing endpoints with M. fortuitumgroup – report as “resistant”
_____________________________CLSI, M24-A2, 2011
48
Quality Assurance for RGMQuality Assurance for RGMSusceptibility TestingSusceptibility Testing
Initial Validation
Susceptibility TestingSusceptibility TestingInitial ValidationQuality ControlProficiency testingProficiency testingParallel Testing (Accredited Labs)Reference StrainsReference Strains
M. peregrinum ATCC 700686S aureus ATCC 29213S. aureus ATCC 29213*P. aeruginosa ATCC 27853E faecalis ATCC 21212
49
E. faecalis ATCC 21212_______CLSI, M24-A2, 2011
Changes to RGM QC Ranges Changes to RGM QC Ranges (µg/mL)(µg/mL)(µg/mL)(µg/mL)
M. peregrinum P. aeruginosa E. faecalis S. aureusDrug ATCC 700686 ATCC 27853* ATCC 29212* ATCC 29213Drug ATCC 700686 ATCC 27853* ATCC 29212* ATCC 29213
Amikacin 1-4 64-256Cefoxitin 4-32Cefoxitin 4 32Ciprofloxacin 0.25-1 0.25-2 Doxycycline 2-8 Imipenem 2-16 1-4 0 5-2Imipenem 2-16 1-4 0.5-2Linezolid 1-8 1-4Meropenem 2-16 0.03-0.12 Minocycline 0 12 0 5 1 4 0 06 0 5Minocycline 0.12-0.5 1-4 0.06-0.5Moxifloxacin < 0.06-0.25 1-8 0.06-0.5 0.015-0.12TMP-SMX <0.25/4.8-2/38 8/152-32/608 <0.5/9.5 ≤0.5/9.5
50
Tobramycin 2-8 0.25-1 8-32________________ CLSI M24-A2, 2011
Slowly Growing NTMSlowly Growing NTMy gy g
M. marinumM. kansasii
M. avium complex (MAC)Other NTMOther NTM
51
M. marinumM. marinumM. marinumM. marinum
• Routine MICs not recommended• All untreated strains have same drug g
pattern • Acquired mutational resistance is rareq• MICs performed at 3 months if still
culture (+)( )_______________________________CLSI, M24-A2, 2011
52
M. marinumM. marinum (cont.)
Clinically Recommended Agents
( )
Clinically Recommended Agents• Clarithromycin
RMP• RMP• Doxycycline/Minocycline• TMP-SMX• RMP + EMB_____________________________CLSI, M24-A2, 2011
53
Broth Microdilution Breakpoints Broth Microdilution Breakpoints forfor Mycobacterium marinumMycobacterium marinumfor for Mycobacterium marinumMycobacterium marinumAntimicrobial Agent Breakpoints
( / L)(µg/mL) Amikacin >32Ciprofloxacin >2pClarithromycin >16Doxycycline/Minocycline >4Ethambutol >4Moxifloxacin >2Rif b ti 2Rifabutin >2Rifampin >1Trimethoprim sulfamethoxazole >2/38
54
Trimethoprim sulfamethoxazole >2/38___ CLSI, M24-A2, 2011
M. kansasiiM. kansasiiM. kansasiiM. kansasii
R ti t ti f if i (RMP) d CLARI l• Routine testing of rifampin (RMP) and CLARI only• RMP susceptibility <1 µg/mL
CLARI <8 g/mL S• CLARI <8 µg/mL = S• Test 2o agents only if RMP resistant (treatment
failure generally seen only with RMP resistance;failure generally seen only with RMP resistance; testing other TB drugs can be problematic)
• If RMP susceptible will be rifabutin susceptibleIf RMP susceptible, will be rifabutin susceptible (HIV patients on protease inhibitors)_________
CLSI M24-A2, 2011
55
Broth MICs Indicating Resistance for Broth MICs Indicating Resistance for M kansasiiM kansasiiM. kansasiiM. kansasii
Antimycobacterial Agents MIC Indicating Resistance(µg/mL)(µg/mL)
Primary AgentsClarithromycin* >16Rif i 1Rifampin >1
Secondary AgentsAmikacin >32Ci fl i /L fl i 2Ciprofloxacin/Levofloxacin >2Ethambutol >4Linezolid* >16Moxifloxacin* >2Rifabutin >2Trimethoprim-Sulfamethoxazole >2/38___________
56
*Changes proposed in CLSI, M24-A2, 2011
M. kansasiiM. kansasiiClinically Recommended AgentsClinically Recommended AgentsClinically Recommended AgentsClinically Recommended Agents
• Clarithromycin, EMB, RMP/RBT• INH EMB RMP/RBTINH, EMB, RMP/RBT
57
M. kansasiiM. kansasii
Summary of ChangesSummary of Changes
• Addition of clarithromycin as Primary AgentAddition of clarithromycin as Primary Agent• Addition of moxifloxacin and linezolid as
secondary agentssecondary agents• Includes MICs indicating resistance to
all agents testedall agents tested___________________________________CLSI M24 A2 2011
58
CLSI, M24-A2, 2011
Quality Control Ranges of MICs for Quality Control Ranges of MICs for Testing of Testing of Mycobacterium kansasiiMycobacterium kansasii to to gg yy
RifampinRifampinOrganism Acceptable Range
(g/mL)M. kansasii ATCC 12478 < 1M. marinum ATCC 927 < 0.25-1E. faecalis ATCC 29212 0.5-4_________________________________________________CLSI M24 A2 2011CLSI, M24-A2, 2011
59
M. aviumM. avium complex (MAC)complex (MAC)M. aviumM. avium complex (MAC)complex (MAC)
Recommendation for which isolates to test:Recommendation for which isolates to test:• Initial isolates to establish baseline value• Isolates from patients on prior macrolideIsolates from patients on prior macrolide
therapy• Isolates from patients who developIsolates from patients who develop
bacteremia on macrolide prophylaxis• Isolates from patients who relapse onIsolates from patients who relapse on
macrolides
60
_____________________________________CLSI, M24-A2, 2011
M. avium M. avium complex (MAC)complex (MAC)p ( )p ( )• Clarithromycin: Class drug for macrolides y g• Susceptibility evaluated on basis of
Clarithromycin MICs onlyy y Broth based system
• No correlation between in vitro susceptibilityNo correlation between in vitro susceptibility results for antituberculous agents (RMP, EMB, RBT) with clinical outcome)
• Repeat MICs at 3 mos (disseminated); 6 mos(chronic respiratory) MAC
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(chronic respiratory) MAC________________CLSI, M24-A2, 2011
MAC: Clarithromycin ResistanceMAC: Clarithromycin ResistanceMAC: Clarithromycin ResistanceMAC: Clarithromycin Resistance
• Untreated strains MICs < 8 µg/mL -responders
• Relapse strains after treatment failureMICs > 32 µg/mL - no longer respond µg g pto macrolides
________________________________CLSI, M24-A2, 2011
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MAC: Acquired (Mutational) MAC: Acquired (Mutational) R i t t Cl ith iR i t t Cl ith iResistance to ClarithromycinResistance to Clarithromycin
• 100% of hi level, ClariR isolates have mutations A2058 or A2059 in 23S rRNA gene
• Untreated strains with intermediate/ resistant MICs are rare
• I/R Untreated strains may indicate y“mixed population”
_______________________________
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_______________________________CLSI, M24-A2, 2011
MAC: Acquired (Mutational) MAC: Acquired (Mutational) R i t t Cl ith iR i t t Cl ith iResistance to Clarithromycin Resistance to Clarithromycin (cont’d)
Closely monitor “I” strains for• Closely monitor “I” strains for development of macrolide resistance
• Azithromycin/Clarithromycin have same RNA t ti (If S t Cl i S t A i &rRNA mutation (If S to Clari, S to Azi &
vice versa)_________________________________CLSI, M24-A2, 2011Meier, et al. J. Infect. Dis. 1996
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Macrolide Resistant MACMacrolide Resistant MAC
Reasonable to test/For interpretation use tentative breakpoints
• MoxifloxacinS G• Linezolid – Same as RGM breakpoints
• No breakpoints established for aminoglycosides (Amikacin Streptomycin)aminoglycosides (Amikacin, Streptomycin)
• No 1st line antituberculous agents should be reportedreported
• Consult expert in treatment of macrolide resistant MAC
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Broth Microdilution MIC Broth Microdilution MIC B k i t (B k i t ( / L) f MAC/ L) f MACBreakpoints (Breakpoints (µg/mL) for MACg/mL) for MAC
Method / Antimicrobial _ MIC_______________Broth Microdilution Susceptible Intermediate Resistant
(pH 7.3-7.4)Cl ith i 1 < 8 16 > 32Clarithromycin1 < 8 16 > 32Moxifloxacin2,3 < 1 2 > 4Linezolid2,3 < 8 16 > 32Linezolid 8 16 32
RadiometricClarithromycin (pH 6.8) < 16 32 > 32Clarithromycin (pH 7.3-7.4) < 4 8-16 ≥ 32
1Primary agent 2Secondary Agent 3Tentative breakpoints__*Changes proposed in CLSI M24 A2 2011
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_______________*Changes proposed in CLSI M24-A2, 2011_________CLSI, M24-A, 2003
Quality Control Ranges of Quality Control Ranges of Broth Microdilution MICs for Broth Microdilution MICs for
Mycobacterium avium Mycobacterium avium ATCC 700898ATCC 700898
Macrolide pH MIC range ( / L)(µg/mL)
Cl ith i 6 8 1 4Clarithromycin 6.8 1-4Clarithromycin 7.3- 7.4 0.5-2_____________________________________CLSI, M24-A2, 2011
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Other Slowly Growing NTMOther Slowly Growing NTMOther Slowly Growing NTMOther Slowly Growing NTM
T f i l t t di d• Too few isolates studied• No specific susceptibility method
recommended• Generally test as for M. kansasiiy
including 2o panel• Must validateMust validate________________________________CLSI, M24-A2, 2011
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Other Slowly Growing NTMOther Slowly Growing NTM
M terrae/nonchromogenicumM. terrae/nonchromogenicumM. simiae M. xenopiM. szulgai M. celatumM lentiflavum M malmoenseM. lentiflavum M. malmoenseNewly described species_______________________________
CLSI, M24-A2, 2011
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Fastidious Species of NTMFastidious Species of NTMpp
No current standardizedNo current standardized susceptibility method
M. haemophilumM. haemophilumRequires hemin/iron compounds
Agar disk elution/"X" strips*Agar disk elution/ X stripsBroth microdilution/ferric ammonium citrate
Extended incubation 2-3 wks 28-30o Cte ded cubat o 3 s 8 30 C*Appendix J has a proposed method
CLSI M24 A2 2011
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CLSI, M24-A2, 2011
Fastidious Species of NTMFastidious Species of NTMpp(cont’d)
No current standardized methodNo current standardized methodNo current standardized method No current standardized method M. genavense
Requires Mycobactin JRequires Mycobactin J supplementationE t d d i b ti 6 kExtended incubation > 6 wks
M. ulceransExtended incubation 4-6 wks
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___________________________CLSI, M24-A2, 2011
Summary: CLSI, M24Summary: CLSI, M24--A2, 2011A2, 2011
RGM
• Recommended susceptibility method: Broth Microdilution
• No antituberculous agents testedI t ti it i f• Interpretive criteria forAmikacin Doxycycline *MinocyclineCefoxitin Imipenem *MoxifloxacinCefoxitin Imipenem MoxifloxacinCiprofloxacin Linezolid *TMP/SMXClarithromycin *Meropenem Tobramycin
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Clarithromycin Meropenem Tobramycin__ *New
Summary: CLSI M24Summary: CLSI M24--A2 2011A2 2011Summary: CLSI, M24Summary: CLSI, M24 A2, 2011A2, 2011Slowly Growing Mycobacteria
• M. marinum – Routine susceptibility not recommendedrecommended
• M. kansasii – RMP and clarithromycin susceptibility only except for RMP resistantsusceptibility only except for RMP resistant isolates
• MAC – Clarithromycin susceptibility only except y p y y pfor moxifloxacin, linezolid (tentative breakpoints)– Broth method recommended and no testing of
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TB agents
Summary: CLSI, M24Summary: CLSI, M24--A2, 2011A2, 2011yy
QC organismsgRGM: M. peregrinum ATCC 700686
S. aureus ATCC 29213*P. aeruginosa ATCC 27853*E. faecalis ATCC 29212
MAC: M. avium ATCC 700898M. kansasii M. kansasii ATCC 12478
M marinum ATCC 927M. marinum ATCC 927E. faecalis ATCC 29212__
*Modification
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Modification
Susceptibility Testing (AST) Susceptibility Testing (AST) C tC tCaveatCaveat
• For labs that encounter NTM infrequently, the recommendation is to refer isolates to an established reference lab for AST
• For labs that elect to do AST, test performance/proficiency must be p p yevaluated initially and maintained regularly
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g y
AcknowledgmentsAcknowledgmentsAcknowledgmentsAcknowledgmentsCLSI Subcommittee on Antimycobacterial
S tibilit T ti G il W d M D Ch iSusceptibility Testing – Gail Woods M.D., Chair
UTHSCT Susceptibility Testing LabUTHSCT Susceptibility Testing LabRichard J. Wallace, Jr., M.D.Kimberly Kriel Ravikiran VasireddyKimberly Kriel Ravikiran VasireddyPamela Newton Paula Johnson Shirley Nichols Ann McClendonShirley Nichols Ann McClendonLinda Bridge Mann Teagen MartinJoanne Woodring
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Joanne Woodring
Aerobic ActinomycetesAerobic ActinomycetesAerobic ActinomycetesAerobic Actinomycetes
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Aerobic ActinomycetesAerobic Actinomycetesyy
• Recommended method: broth microdilution• Recommended method: broth microdilution– For R. equi, use microtiter dilution panels for gram-
positive aerobic bacteria & follow guidelines in M7-A8p g• Potential problems requiring further studies:
– Ceftriaxone endpoints difficult to interpret consistently– SXT endpoints difficult to interpret consistently
• Supplement with disk diffusion using sulfisoxazole disk
False resistant results for ceftriaxone & N brasiliensis– False-resistant results for ceftriaxone & N. brasiliensis– False-resistant results for imipenem & N. farcinica
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Aerobic ActinomycetesAerobic Actinomycetes
• Antimicrobial agents: new in M24-A2
yy
Antimicrobial agents: new in M24 A2– For R. equi only, include rifampin & vancomycin
• Inoculum suspension– Prepare heavy suspension in sterile, deionized water or
saline from growth on blood or trypticase soy agarBreak up clumps using micropestle or vortexing with– Break up clumps using micropestle or vortexing with glass beads
– Allow clumps to settle (about 15 minutes)p ( )– Add supernatant to 2ml water or saline; check turbidity
with nephelometer to = density of 0.5 McFarland
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Aerobic ActinomycetesAerobic Actinomycetes
• Inoculate tray cover with adhesive seal & place
yy
• Inoculate tray, cover with adhesive seal, & place in plastic bag
• Incubation: 35±2C, ambient air,• Read at 72 hr (R. equi, 24 hr; Tsukamurella, 24-
48 hr)– If growth <2+, incubate & read daily for up to 5 days
• MIC=lowest concentration that inhibits visible growth except for SXTgrowth, except for SXT
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Reading SXT EndReading SXT End--PointPointgg
• General recommendation: well with 80% inhibition• General recommendation: well with 80% inhibition of growth compared with growth in + control well or well with lowest SXT concentration (if more (than + control)
• More practical: dilution showing significant difference in amount of growth compared with + control well or to an adjacent well with a highercontrol well or to an adjacent well with a higher drug concentration
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SulfisoxazoleSulfisoxazole Disk DiffusionDisk Diffusion
• 0 5 McFarland suspension• 0.5 McFarland suspension• Follow guidelines in CLSI M02• 250 µg diskµg• Incubate in ambient air, 35+/-2C, 72 hr• Evaluate growth: should not be confluent; streak
marks should be obvious with clear areas between streaks apparent
• Compare to SXT MIC if latter is questionable• Compare to SXT MIC if latter is questionable
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SulfisoxazoleSulfisoxazole Disk Diffusion*Disk Diffusion*
• Zone ≥ 35mm = Susceptible• Zone ≤ 15mm = ResistantZone 15mm Resistant• Zone 16-34 mm uninterpretable (insufficient data)• If disk diffusion and MIC results are discrepant,
retest or send to reference lab
*From Wallace et al. Disk diffusion testing of Nocardia species. JIDFrom Wallace et al. Disk diffusion testing of Nocardia species. JID 1977;135:568.
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Reporting ResultsReporting Resultsp gp g
• Nocardia: report MIC value & interpretation• Nocardia: report MIC value & interpretation– Compare to those expected for the species (Appendix K)– If differ from expected, repeat &/or send to reference lab p , p
with expertise– Report sulfa-R with caution (most species susceptible)
R i t MIC l & i t t ti i• R. equi: report MIC value & interpretation using breakpoints for S. aureus (M100)– Tentative pending more dataTentative pending more data
• Other genera: report MIC value, referring to footnote “a” in Table 9
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Table 9, Footnote aTable 9, Footnote a,,
• Breakpoints in this table apply to Nocardia• Breakpoints in this table apply to Nocardia and can tentatively be used for other aerobic actinomycetes Theseaerobic actinomycetes…..These breakpoints are considered tentative and should be reported as such pending theshould be reported as such pending the accumulation of further information.
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Aerobic Actinomycetes: QCAerobic Actinomycetes: QCyy
• Test appropriate strains weekly or each day• Test appropriate strains weekly or each day test is performed (if less than weekly)
S ATCC 29213– S. aureus ATCC 29213– P. aeruginosa ATCC 27853
E li ATCC 35218 (f i illi l l i– E. coli ATCC 35218 (for amoxicillin-clavulanic acid)
• Acceptable ranges for strains in M100• Acceptable ranges for strains in M100
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??? QUESTIONS ?????? QUESTIONS ???
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