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Dr.T.V.Rao MD<br />EXTENDED SPECTRUM B-LACTAMASES(esbl)basis, detection and reporting<br />Dr.T.V.Rao MD<br />1<br />
V. cholera<br />C. jejuni<br />Helicobacter pylori<br />Acinetobacter spp.<br />Gram Negative Bacilli <br />Stenotrophomon...
History of resistance in gram-negative bacteria<br />1950<br />1960<br />1970<br />1980<br />1990<br />2000<br />1965 <br ...
Evolution of -Lactamases<br />Plasmid-mediated TEM and SHV -lactamases<br />Extended-spectrum<br />Cephalosporins<br />A...
Committees controlling resistance patterns<br />BSAC<br />United Kingdom<br />SRGA<br />Sweden<br />CA-SFM<br />France<br ...
Standardisation<br />1959  Ericsson & Steers – evaluation of methods<br />1961  WHO – standardisation<br />1964  Isenberg ...
Growing incidence of Esbl producers<br />Dr.T.V.Rao MD<br />7<br />
β-lactamases classification<br />Molecular class:<br />A:<br />TEM<br />SHV<br />other<br />B: <br />Metalloenzymes (carba...
Types of β-lactamases<br />ESBLs <br />TEM related<br />SHV related<br />OXA related<br />CTX-M <br />Other<br />ampCβ-lac...
Extended-Spectrum -lactamases ESBLs<br />Mutations of TEM-1, TEM-2, SHV-1<br />Inactivate -lactams with an oxyimino grou...
Broad spectrum b-lactamase (blaTEM)<br />ESBL<br />(TEM related)<br />&<br />&<br />Mutation<br />Plasmid<br />transfer<br...
H<br />H<br />S<br />C<br />R-CONH<br />C<br />CH3<br />C<br />N<br />CH3<br />O<br />COOH<br />-Lactamase Activity<br />...
-Lactamase Activity<br />H<br />H<br />S<br />C<br />R-CONH<br />C<br />CH3<br />C<br />N<br />O<br />CH3<br />H<br />O<b...
L<br /><br /><br /><br /><br /><br />L<br /><br /><br />L<br /><br /><br /><br /><br /><br /><br /><br />...
The term ESBLs is used to mean acquired, class A β-lactamases that hydrolyze and confer resistance to oxyimino- ‘2nd- and ...
ESBL<br />Confer resistance to 1st , 2nd, 3rd cephalosporins. <br />Most are susceptible to β-lactamase inhibitors<br />Mo...
Further complicating matters:<br />More than one gene of β-lactamase / ESBL / ampC / carbapenemase can be carried on the s...
ESBLs include:<br />Cephalosporin-hydrolysing mutants of TEM and SHV - the common plasmid-mediated penicillinases of Enter...
ESBLs are not the sole β-lactamases to confer resistance to 2nd and 3rd generation cephalosporins, but are the most import...
They should be distinguishedfrom other important modes of resistance to 2nd and 3rd generation cephalosporins, eg:<br />Hy...
LABORATORY DETECTION: SCREENING andCONFIRMATION<br />Dr.T.V.Rao MD<br />21<br />
The basic strategy to detect ESBL producers is to use an indicator cephalosporin to screen for likely producers, then to s...
SCREENING<br />The ideal indicator cephalosporin is one to which all ESBLs confer resistance, even when their production i...
Dr.T.V.Rao MD<br />24<br />
Detection Strategy: step 1<br />Screen Enterobacteriaceae with :<br /><ul><li>Cefpodoxime- best general ESBL substrate
Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively</li></ul>Spread of CTX-M into community means ...
Detection of ESBLs: step 2<br />Seek ceph/clav synergy in ceph R isolates <br />Double disc<br />Combination disc<br />Ete...
ESBL detection<br />2 Steps:<br /><ul><li>Screen cefpodoxime ; cefotaxime & ceftazidime
Synergy test with ceph/clavulanic acid</li></ul>Combination discs are most cost effective synergy tests; Etest a good alte...
ESBL Confirmatory Tests<br /> Double-disk synergy (DDS) test<br /><ul><li> CAZ and CAZ/CA disks
 CTX and CTXCA disks
 Confirmatory testing </li></ul>  requires using both CAZ <br />  and CTX alone and with CA<br /><ul><li> 5 mm enhancement...
Dr.T.V.Rao MD<br />29<br />
Dr.T.V.Rao MD<br />30<br />
ESBL Confirmation<br />		       Clavulanate Inhibition with<br />Cefotaxime		-		+		+		-<br />Ceftazidime	-		+		-		+<br />N...
CAZ+CA<br />CTX+CA<br />Test for<br />E. coli <br />K. pneumoniae<br />K. oxytoca<br />CMZ<br />CTX<br />CAZ<br />Dr.T.V.R...
ESBL Confirmatory Test<br />Positive for ESBL<br />Cefotax/CA<br />Ceftaz/CA<br />Ceftaz<br />Cefotax<br />33<br />Dr.T.V....
34<br />ESBL Confirmatory Test Negative for ESBL <br />Ceftaz/CA<br />Cefotaxime/CA<br />Ceftaz<br />Cefotax<br />34<br />...
Dr.T.V.Rao MD<br />35<br />
Dr.T.V.Rao MD<br />36<br />
Dr.T.V.Rao MD<br />37<br />
Dr.T.V.Rao MD<br />38<br />
ESBL Example<br />						MIC (g/ml)<br />Cefotaxime					>32<br />Ceftazidime				0.5<br />Cefotaxime/clavulanate		        ...
How to ReportKlebsiella pneumoniae (ESBL)<br />Cefepime	    S (?) R<br />Gentamicin	    R<br />Amikacin	    S<br />Imipe...
It follows that the logical indicator is either cefpodoxime or BOTH of cefotaxime and ceftazidime resistance.<br />An alte...
AmpC<br />Gene on chromosome<br />Produced by virtually all GNBs<br />Activity generally low<br />NOT inhibited by -lacta...
AmpC -lactamaseE. coli/Klebsiella spp.-Typical<br />Chromosomal<br />Produced in minimal amount<br />NOT inducible<br />D...
AmpC -lactamaseE. coli/Klebsiella spp.- Genetic Changes<br />AmpC genes mutation<br />Low amount-destroy ampicillin,     ...
AmpC -lactamaseOther GNBs-Typical<br />Chromosomal<br />Produced in small amount<br />Inducible (hyper production)<br />R...
Etest for ESBLs<br />Cefotaxime<br />Cefotaxime<br />+<br />clavulanate<br />Dr.T.V.Rao MD<br />46<br />
Etest for ESBLs<br />Cefotaxime<br />Cefotaxime<br />+<br />clavulanate<br />Dr.T.V.Rao MD<br />47<br />
Dr.T.V.Rao MD<br />48<br />Quality control in                 esbl detection<br />
Positive controls should be used to ensure the performance of ESBL confirmatory tests. Three ESBL-positive E. coli strains...
The CLSI recommends K. pneumoniae ATCC 700603 as an ESBL-producing QC control, as does AB Biodisk (Etest). This strain may...
Zones of the cephalosporin and cephalosporin + clavulanate discs for ESBL-negative E. coli should be equal or, at worst, w...
Briefly, revising breakpoints involves systematic review of microbiological, pharmacologic, and clinical data. Recognized ...
Follow the New Guidelines CLSI 2010<br />Guidelines for cephalospins for Enterobacteriaceae in accordance with the 2010 Cl...
Why do breakpoints sometimes need to be revised? <br />Breakpoints need to be revised due to changing resistance mechanism...
Enterobacteriaceae:  Revised Breakpoints for Cephalosporins  <br />Dr.T.V.Rao MD<br />55<br />
Agent     Old (M100-S19) Revised (M100-S20) <br />S   I R  S  I R <br />Cefazolin 	≥18 	15-17 	≤14 	NA NA 	NA 	<br />Cefot...
Agent         M100-S19 M100-S20 <br />S  I  RS  I   R<br />Cefuroxime	≤8 	16  ≥32 	≤8 	 16 	≥32 	<br />Cefepime 	≤8 	16 	≥...
Dr.T.V.Rao MD<br />58<br />Why were the breakpoints for cefepime and cefuroxime (parenteral) not revised? <br />The cefepi...
Dr.T.V.Rao MD<br />59<br />Why are there no disk diffusion breakpoints for Cefazolin?<br />Studies have not yet been compl...
Bacteria not to test for ESBL’s<br />Acinetobacter<br />Acinetobacter often S to clavulanate alone<br />S. maltophilia<br ...
Pitfalls in ESBL detection<br /><ul><li>Methods optimised for E. coli & Klebsiella
More difficult with Enterobacter
clavulanate induces AmpC; hides ESBL
Best advice is to do synergy test (NOT SCREEN) with 4th gen ceph</li></ul>Dr.T.V.Rao MD<br />61<br />
Molecular detection: where and why ?<br /><ul><li>In the Reference Laboratory
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Laboratory detection of ESBL

Laboratory detection of ESBL

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Laboratory detection of ESBL

  1. 1. Dr.T.V.Rao MD<br />EXTENDED SPECTRUM B-LACTAMASES(esbl)basis, detection and reporting<br />Dr.T.V.Rao MD<br />1<br />
  2. 2. V. cholera<br />C. jejuni<br />Helicobacter pylori<br />Acinetobacter spp.<br />Gram Negative Bacilli <br />Stenotrophomonas maltophilia<br />Many other <br />(H. influenza, etc..)<br />Pseudomonas aeruginosa<br />Enterobacteriaceae<br />Dr.T.V.Rao MD<br />2<br />
  3. 3. History of resistance in gram-negative bacteria<br />1950<br />1960<br />1970<br />1980<br />1990<br />2000<br />1965 <br />Broad spectrum β–lactamases (TEM-1 in E. coli)<br />ESBL outbreaks in France<br />1940 Penicillinase detected in E. coli<br />1983 Extended spectrum β-lactamases<br />TEM-1 widespread<br />Carbapenemases<br />1964<br />Cephalothin use<br />1941<br />Penicillin use<br />Early 1980s<br />3rd generation ceph.<br />1959<br />β-lactamase resistant penicillin's: Methicillin<br />1985<br />Carbapenems (Imipenem)<br />1960s<br />Broad spectrum/ extended spectrum penicillin's<br />1976<br />β–lactamases inhibitors<br />2005 <br />Tigecycline<br />1928<br />Fleming<br />Dr.T.V.Rao MD<br />3<br />
  4. 4. Evolution of -Lactamases<br />Plasmid-mediated TEM and SHV -lactamases<br />Extended-spectrum<br />Cephalosporins<br />Ampicillin<br />1983<br />1965<br />1970s<br />1988<br />2000<br />1963<br />Look and you will find ESBL<br />TEM-1<br />E.coli<br />S.paratyphi<br />TEM-1<br />Reported in <br />28 Gm(-) sp<br />ESBL in <br />Europe<br />ESBL <br />in USA<br />> 130 ESBLs<br />Worldwide<br />Dr.T.V.Rao MD<br />4<br />
  5. 5. Committees controlling resistance patterns<br />BSAC<br />United Kingdom<br />SRGA<br />Sweden<br />CA-SFM<br />France<br />CRG<br />Netherlands<br />DIN<br />Germany<br />NWGA<br />Norway<br />CLSI (NCCLS) USA<br />Dr.T.V.Rao MD<br />5<br />
  6. 6. Standardisation<br />1959 Ericsson & Steers – evaluation of methods<br />1961 WHO – standardisation<br />1964 Isenberg – comparison of methods in USA<br />1964 Truant – standardised tube dilution MICs<br />1966 Bauer-Kirby<br />1975 NCCLS  CLSI<br />1998 BSAC Standardised Method <br />2009 EUCAST Standardised Method<br />Dr.T.V.Rao MD<br />6<br />
  7. 7. Growing incidence of Esbl producers<br />Dr.T.V.Rao MD<br />7<br />
  8. 8. β-lactamases classification<br />Molecular class:<br />A:<br />TEM<br />SHV<br />other<br />B: <br />Metalloenzymes (carbapenemases)<br />C: <br />Prototype: chromosomal ampC<br />D: <br />OXA (oxacillin hydrolyzing enzymes)<br />Enzyme type (by substrate profile):<br />Penicillinase<br />Broad-spectrum<br />Extended Spectrum<br />Carbapenemase<br />Genetic classification:<br />plasmids mediated<br />Chromosomal<br />http://www.lahey.org/studies/webt.asp<br />Dr.T.V.Rao MD<br />8<br />
  9. 9. Types of β-lactamases<br />ESBLs <br />TEM related<br />SHV related<br />OXA related<br />CTX-M <br />Other<br />ampCβ-lactamases<br />Resistant to β-lactamase inhibitors<br />chromosomal<br />Carbapenemases<br />Metallo- β-lactamases<br />Serine carbapenemases<br />β-lactamases<br />Penicillinase: gene blaZ , inducible, on transposon (can move between chromosome and plasmid).<br />Broad spectrum β-lactamases <br />(plasmid encoded)<br />TEM<br /> SHV <br /> OXA (mainly in pseudomonas)<br />Dr.T.V.Rao MD<br />9<br />
  10. 10. Extended-Spectrum -lactamases ESBLs<br />Mutations of TEM-1, TEM-2, SHV-1<br />Inactivate -lactams with an oxyimino group (third-generation cephalosporins and aztreonam)<br />Plasmid-mediated (often other R genes)<br />70 TEM and 15 SHV types<br />Dr.T.V.Rao MD<br />10<br />
  11. 11. Broad spectrum b-lactamase (blaTEM)<br />ESBL<br />(TEM related)<br />&<br />&<br />Mutation<br />Plasmid<br />transfer<br />Genetic Mechanism <br />Penicillinase blaZ<br />Transformation<br />Dr.T.V.Rao MD<br />11<br />
  12. 12. H<br />H<br />S<br />C<br />R-CONH<br />C<br />CH3<br />C<br />N<br />CH3<br />O<br />COOH<br />-Lactamase Activity<br />-lactam<br />Enzyme-Ser-OH<br />Dr.T.V.Rao MD<br />12<br />
  13. 13. -Lactamase Activity<br />H<br />H<br />S<br />C<br />R-CONH<br />C<br />CH3<br />C<br />N<br />O<br />CH3<br />H<br />O<br />COOH<br />HOH<br />Ser<br />Enzyme<br />Dr.T.V.Rao MD<br />13<br />
  14. 14. L<br /><br /><br /><br /><br /><br />L<br /><br /><br />L<br /><br /><br /><br /><br /><br /><br /><br /><br />L<br /><br /><br /><br /><br />L<br />L<br /><br /><br /><br /><br />L<br /><br /><br /><br />b-lactamase<br />production<br />L<br /><br />L<br /><br />Dr.T.V.Rao MD<br />14<br />
  15. 15. The term ESBLs is used to mean acquired, class A β-lactamases that hydrolyze and confer resistance to oxyimino- ‘2nd- and 3rd-generation’ cephalosporins, eg cefuroxime, cefotaxime, ceftazidime and ceftriaxone<br />What are ESBL producing bacteria.<br />Dr.T.V.Rao MD<br />15<br />
  16. 16. ESBL<br />Confer resistance to 1st , 2nd, 3rd cephalosporins. <br />Most are susceptible to β-lactamase inhibitors<br />Most are susceptible to 4th cephalosporins<br />All are susceptible to carbapenems<br />Diversity of ESBL<br />SHV (widespread)<br />TEM (>100 types)<br />OXA <br />Predominantly in Pseudomonas<br />less susceptible to β-lactamase inhibitors<br />CTX-M<br />Probably independent evolution<br />Highly resistant to 3rd generation cephalosporins<br />initially in South America, Far East & Eastern Europe<br />Probably most frequent worldwide<br />Clonal spread has been documented<br />Dr.T.V.Rao MD<br />16<br />
  17. 17. Further complicating matters:<br />More than one gene of β-lactamase / ESBL / ampC / carbapenemase can be carried on the same plasmid.<br />Genes of ESBL are carried on plasmids that usually carry additional resistant genes: frequently MDR<br />Laboratory diagnosis confusing: <br /> susceptibility profiles sometimes <br /> misleading: “hidden resistance” -> <br /> CLSI guidelines are changing.<br />CTX-M clones appearing in the <br /> community (Canada, Greece, Spain,<br /> Italy).<br />Dr.T.V.Rao MD<br />17<br />
  18. 18. ESBLs include:<br />Cephalosporin-hydrolysing mutants of TEM and SHV - the common plasmid-mediated penicillinases of Enterobacteriaceae. Well over 100 such variants are known <br />• CTX-M types. These evolved separately, at least some of them via the escape and mutation of chromosomal β-lactamases of Kluyvera species. Over 30 variants are known Obscure types, e.g. VEB and PER, not yet of concern in the UK; also OXA (Class D) ESBLs from Pseudomonas aeruginosa, in Turkey.<br />Dr.T.V.Rao MD<br />18<br />
  19. 19. ESBLs are not the sole β-lactamases to confer resistance to 2nd and 3rd generation cephalosporins, but are the most important. They occur mostly in Enterobacteriaceae (e.g. E. coli, Klebsiella species and Enterobacter species) and rarely in non-fermenters (e.g. P. aeruginosa).<br />Esbl are important cause of resistance <br />Dr.T.V.Rao MD<br />19<br />
  20. 20. They should be distinguishedfrom other important modes of resistance to 2nd and 3rd generation cephalosporins, eg:<br />Hyper produced chromosomal AmpC β-lactamases, especially in Enterobacter species.<br />• Plasmid-mediated AmpC β-lactamases, in Klebsiella spp. and E. coli (rare)<br />• Hyper produced K1 chromosomal β-lactamases in K. oxytoca not pneumoniae)<br />• Efflux-mediated resistance in P. aeruginosa<br />• Various ill-defined mechanisms in Acinetobacter species.<br />Dr.T.V.Rao MD<br />20<br />
  21. 21. LABORATORY DETECTION: SCREENING andCONFIRMATION<br />Dr.T.V.Rao MD<br />21<br />
  22. 22. The basic strategy to detect ESBL producers is to use an indicator cephalosporin to screen for likely producers, then to seek cephalosporin/clavulanate synergy, which distinguishes ESBL producers from, for example, strains that hyper producer AmpC or K1 enzymes.<br />Primary testing for Esbl detection<br />Dr.T.V.Rao MD<br />22<br />
  23. 23. SCREENING<br />The ideal indicator cephalosporin is one to which all ESBLs confer resistance, even when their production is scanty. Choice is predicated by the following general traits:<br />TEM and SHV ESBLs – obvious resistance to ceftazidime, variable to cefotaxime<br />CTX-M ESBLs – obvious resistance to cefotaxime: variable to ceftazidime<br />All ESBLs – obvious resistance to cefpodoxime<br />Dr.T.V.Rao MD<br />23<br />
  24. 24. Dr.T.V.Rao MD<br />24<br />
  25. 25. Detection Strategy: step 1<br />Screen Enterobacteriaceae with :<br /><ul><li>Cefpodoxime- best general ESBL substrate
  26. 26. Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively</li></ul>Spread of CTX-M into community means screening must be wider than before<br />See http://www.hpa.org.uk<br />Dr.T.V.Rao MD<br />25<br />
  27. 27. Detection of ESBLs: step 2<br />Seek ceph/clav synergy in ceph R isolates <br />Double disc<br />Combination disc<br />Etest<br />See http://www.hpa.org.uk<br />Dr.T.V.Rao MD<br />26<br />
  28. 28. ESBL detection<br />2 Steps:<br /><ul><li>Screen cefpodoxime ; cefotaxime & ceftazidime
  29. 29. Synergy test with ceph/clavulanic acid</li></ul>Combination discs are most cost effective synergy tests; Etest a good alternative.. or automate<br />Dr.T.V.Rao MD<br />27<br />
  30. 30. ESBL Confirmatory Tests<br /> Double-disk synergy (DDS) test<br /><ul><li> CAZ and CAZ/CA disks
  31. 31. CTX and CTXCA disks
  32. 32. Confirmatory testing </li></ul> requires using both CAZ <br /> and CTX alone and with CA<br /><ul><li> 5 mm enhancement of the inhibition</li></ul> zone of antibiotic/CA combination vs antibiotic <br /> tested alone = ESBL <br />Dr.T.V.Rao MD<br />28<br />
  33. 33. Dr.T.V.Rao MD<br />29<br />
  34. 34. Dr.T.V.Rao MD<br />30<br />
  35. 35. ESBL Confirmation<br /> Clavulanate Inhibition with<br />Cefotaxime - + + -<br />Ceftazidime - + - +<br />NOT ESBL<br />report results as they appear<br />ESBL<br />report cephalosporins, penicillins, <br />aztreonam as “R”<br />Dr.T.V.Rao MD<br />31<br />
  36. 36. CAZ+CA<br />CTX+CA<br />Test for<br />E. coli <br />K. pneumoniae<br />K. oxytoca<br />CMZ<br />CTX<br />CAZ<br />Dr.T.V.Rao MD<br />32<br />
  37. 37. ESBL Confirmatory Test<br />Positive for ESBL<br />Cefotax/CA<br />Ceftaz/CA<br />Ceftaz<br />Cefotax<br />33<br />Dr.T.V.Rao MD<br />33<br />
  38. 38. 34<br />ESBL Confirmatory Test Negative for ESBL <br />Ceftaz/CA<br />Cefotaxime/CA<br />Ceftaz<br />Cefotax<br />34<br />Dr.T.V.Rao MD<br />
  39. 39. Dr.T.V.Rao MD<br />35<br />
  40. 40. Dr.T.V.Rao MD<br />36<br />
  41. 41. Dr.T.V.Rao MD<br />37<br />
  42. 42. Dr.T.V.Rao MD<br />38<br />
  43. 43. ESBL Example<br /> MIC (g/ml)<br />Cefotaxime >32<br />Ceftazidime 0.5<br />Cefotaxime/clavulanate 0.5<br />Ceftazidime/clavulanate 1.0<br />Dr.T.V.Rao MD<br />39<br />
  44. 44. How to ReportKlebsiella pneumoniae (ESBL)<br />Cefepime S (?) R<br />Gentamicin R<br />Amikacin S<br />Imipenem S <br />Ciprofloxacin S<br />Flomoxef S ?<br />Ampicillin R <br />Amoxicillin/clavulanate S<br />Cefazolin R<br />Cefotiam R<br />Cefmetazole S<br />Cefotaxime I R<br />Aztreonam R<br />, NCCLS not recommended<br />Dr.T.V.Rao MD<br />40<br />
  45. 45. It follows that the logical indicator is either cefpodoxime or BOTH of cefotaxime and ceftazidime resistance.<br />An alternative strategy has been proposed for community urines: testing cephalexin or cephradine as the indicator drug, then doing confirmatory ESBL tests on all isolates that are found resistant (these include e.g. all Enterobacter species. and some hyper producers of classical TEM, as well as the ESBL producers). This is not recommended, as some CTX-M-15 producers, <br />Dr.T.V.Rao MD<br />41<br />
  46. 46. AmpC<br />Gene on chromosome<br />Produced by virtually all GNBs<br />Activity generally low<br />NOT inhibited by -lactamase inhibitors<br />Differ in E. coli/Klebsiella versus other GNBs<br />Dr.T.V.Rao MD<br />42<br />
  47. 47. AmpC -lactamaseE. coli/Klebsiella spp.-Typical<br />Chromosomal<br />Produced in minimal amount<br />NOT inducible<br />Dr.T.V.Rao MD<br />43<br />
  48. 48. AmpC -lactamaseE. coli/Klebsiella spp.- Genetic Changes<br />AmpC genes mutation<br />Low amount-destroy ampicillin, 1st cephalosporins<br />High amount-destroy expanded spectrum -lactams<br />Transfer of ampC to plasmid<br />Hyper production<br />Resistant to 3rd cephalosporins, cephamycins<br />Dr.T.V.Rao MD<br />44<br />
  49. 49. AmpC -lactamaseOther GNBs-Typical<br />Chromosomal<br />Produced in small amount<br />Inducible (hyper production)<br />Reversible<br />Dr.T.V.Rao MD<br />45<br />
  50. 50. Etest for ESBLs<br />Cefotaxime<br />Cefotaxime<br />+<br />clavulanate<br />Dr.T.V.Rao MD<br />46<br />
  51. 51. Etest for ESBLs<br />Cefotaxime<br />Cefotaxime<br />+<br />clavulanate<br />Dr.T.V.Rao MD<br />47<br />
  52. 52. Dr.T.V.Rao MD<br />48<br />Quality control in esbl detection<br />
  53. 53. Positive controls should be used to ensure the performance of ESBL confirmatory tests. Three ESBL-positive E. coli strains are available from the NCTC: • CTX-M-15 (cefotaximase) NCTC 13353 TEM-3 (broad-spectrum) NCTC 13351 TEM-10 (ceftazidimase) NCTC 13352<br />CONTROLS FOR ESBL TESTS<br />Dr.T.V.Rao MD<br />49<br />
  54. 54. The CLSI recommends K. pneumoniae ATCC 700603 as an ESBL-producing QC control, as does AB Biodisk (Etest). This strain may be sourced from the ATCC.<br />Either E. coli NCTC 10418 or ATCC 25922 should also be used as a negative control in ESBL confirmation tests. Use of such controls is especially important when the cephalosporin and cephalosporin + clavulanate combination discs are from different batches, which may vary in original content or retained potency.<br />Clsi recommends <br />Dr.T.V.Rao MD<br />50<br />
  55. 55. Zones of the cephalosporin and cephalosporin + clavulanate discs for ESBL-negative E. coli should be equal or, at worst, within + 2 mm. Any greater difference implies malfunction or deterioration.<br />Zone determination guides … <br />Dr.T.V.Rao MD<br />51<br />
  56. 56. Briefly, revising breakpoints involves systematic review of microbiological, pharmacologic, and clinical data. Recognized experts, sponsors (pharmaceutical industry), and regulators participate in the process which includes discussions at public meetings of the CLSI Subcommittee on Antimicrobial Susceptibility Testing that take place twice a year. When establishing original breakpoints for new agents, controlled clinical trial data are required <br />Role of CLSI in Revising Breakpoints in Antibiotic Resistance<br />Dr.T.V.Rao MD<br />52<br />
  57. 57. Follow the New Guidelines CLSI 2010<br />Guidelines for cephalospins for Enterobacteriaceae in accordance with the 2010 Clinical Laboratory Standards Institute (CLSI) recommendations. The following changes will be made to comply with the CLSI.<br />Dr.T.V.Rao MD<br />53<br />
  58. 58. Why do breakpoints sometimes need to be revised? <br />Breakpoints need to be revised due to changing resistance mechanisms and bacterial population distributions, changing science leading to a better understanding of the pharmacologic determinants of clinical response, and adoption of “best practices” by clinicians. <br />Dr.T.V.Rao MD<br />54<br />
  59. 59. Enterobacteriaceae: Revised Breakpoints for Cephalosporins <br />Dr.T.V.Rao MD<br />55<br />
  60. 60. Agent Old (M100-S19) Revised (M100-S20) <br />S I R S I R <br />Cefazolin ≥18 15-17 ≤14 NA NA NA <br />Cefotaxime ≥23 15-22 ≤14 ≥26 23-25 ≤22 <br />Ceftizoxime ≥20 15-19 ≤14 ≥25 22-24 ≤21 <br />Ceftriaxone ≥21 14-20 ≤13 ≥23 20-22 ≤19 <br />Ceftazidime ≥18 15-17 ≤14 ≥21 18-20 ≤17 <br />Aztreonam ≥22 16-21 ≤15 ≥21 18-20 ≤17 <br />S – susceptible<br />I – Intermediate<br />R – Resistant.<br />Disk diffusion breakpoints (mm): <br />Dr.T.V.Rao MD<br />56<br />
  61. 61. Agent M100-S19 M100-S20 <br />S I RS I R<br />Cefuroxime ≤8 16 ≥32 ≤8 16 ≥32 <br />Cefepime ≤8 16 ≥32 ≤8 16 ≥32 <br />Cefotetan ≤16 32 ≥64 ≤16 32 ≥64 <br />Cefoxitin ≤8 16 ≥32 ≤8 16 ≥32 <br />S – susceptible<br />I – Intermediate<br />R – Resistant<br />Following MIC breakpoints were reevaluated for Enterobacteriaceae but were not revised <br />Dr.T.V.Rao MD<br />57<br />
  62. 62. Dr.T.V.Rao MD<br />58<br />Why were the breakpoints for cefepime and cefuroxime (parenteral) not revised? <br />The cefepime breakpoints were not revised based upon clinical trial data and PK-PD evaluations. The clinical trial data showed cefepime efficacy for patients infected with isolates that tested cefepime susceptible (MIC ≤8 μg/ml), but produced an ESBL <br />
  63. 63. Dr.T.V.Rao MD<br />59<br />Why are there no disk diffusion breakpoints for Cefazolin?<br />Studies have not yet been completed to identify the zone diameter breakpoints that correlate with the revised MIC breakpoints for Cefazolin. Initial studies did not reveal clear zone diameter breakpoints and disk diffusion testing of Cefazolin may require a new disk with alternate disk content. <br />
  64. 64. Bacteria not to test for ESBL’s<br />Acinetobacter<br />Acinetobacter often S to clavulanate alone<br />S. maltophilia<br />You get +ve results via inhibition of L-2 chromosomal b-lactamase, which is ubiquitous in the species<br />Dr.T.V.Rao MD<br />60<br />
  65. 65. Pitfalls in ESBL detection<br /><ul><li>Methods optimised for E. coli & Klebsiella
  66. 66. More difficult with Enterobacter
  67. 67. clavulanate induces AmpC; hides ESBL
  68. 68. Best advice is to do synergy test (NOT SCREEN) with 4th gen ceph</li></ul>Dr.T.V.Rao MD<br />61<br />
  69. 69. Molecular detection: where and why ?<br /><ul><li>In the Reference Laboratory
  70. 70. confirmation of unusual resistance
  71. 71. surveillance of resistance mechanisms
  72. 72. monitoring spread of resistance genes / strains
  73. 73. identify strains likely to contain novel resistance mechanisms
  74. 74. In the clinical diagnostic laboratory
  75. 75. rapid detection for patient management
  76. 76. infection control</li></ul>Dr.T.V.Rao MD<br />62<br />
  77. 77. Molecular detection: different needs<br /><ul><li>In the Reference Laboratory,
  78. 78. testing “pure” cultures
  79. 79. myriad assays and formats
  80. 80. numerous bug-drug combinations
  81. 81. In the clinical diagnostic laboratory
  82. 82. directly from specimens
  83. 83. need to target key species
  84. 84. format must be simple, rapid and cost-effective
  85. 85. problems with genes in commensals</li></ul>Dr.T.V.Rao MD<br />63<br />
  86. 86. Simple and multiplex PCR<br />Real-time PCR<br />DNA sequencing<br />Hybridisation-based techniques<br />Molecular detection<br />Dr.T.V.Rao MD<br />64<br />
  87. 87. Molecular tests in clinical labs<br />Simple sample<br />preparation<br />Black box <br />approach: <br />molecular biology <br />steps hidden<br />Simple end-product <br />detection<br /><ul><li> Must be rapid (TATs), inexpensive, reliable !
  88. 88. Platform must be sufficiently versatile to justify investment
  89. 89. Relatively hands-free, with scope for automation
  90. 90. On-going – e.g. <30 min test for ESBL detection</li></ul>Dr.T.V.Rao MD<br />65<br />
  91. 91. Chips with everything…<br />going beyond AST!<br />Total profiling; <br />more cost-effective than PCR<br /><ul><li> species identification
  92. 92. resistance genes
  93. 93. virulence genes
  94. 94. epidemicity predictors
  95. 95. strain-specific markers</li></ul>Dr.T.V.Rao MD<br />66<br />
  96. 96. Molecular detection: the inherent problem<br /><ul><li>Molecular methods only detect known mechanisms
  97. 97. only as good as available sequence data
  98. 98. resistant isolates with known genes identified
  99. 99. & new variants, if sufficient homology
  100. 100. false-resistance (unexpressed / partial genes)
  101. 101. Susceptibility must always be confirmed
  102. 102. can’t base treatment on a negative molecular result
  103. 103. can’t detect genuinely new resistance mechanisms
  104. 104. will never (?) replace cheap phenotypic methods</li></ul>Dr.T.V.Rao MD<br />67<br />
  105. 105. Hand washing can reduce the prevalence and spread of esbl producers<br />Dr.T.V.Rao MD<br />68<br />
  106. 106. Dr.T.V.Rao MD<br />69<br />Follow me for Articles of Interest on infectious diseases and Microbiology ..<br />
  107. 107. Dr.T.V.Rao MD<br />70<br />Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical Professionals in the Development World<br />Email<br />doctortvrao@gmail.com<br />

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Laboratory detection of ESBL

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