laboratory detection of esbl
DESCRIPTION
Laboratory detection of ESBLTRANSCRIPT
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DR.T.V.RAO MD 1
Dr.T.V.Rao MD
EXTENDED SPECTRUM
B-LACTAMASES(ESBL)
BASIS, DETECTION AND REPORTING
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DR.T.V.RAO MD 2
Gram Negative Bacilli
V. choleraC. jejuni
Helicobacter pylori
EnterobacteriaceaePseudomonas
aeruginosa
Stenotrophomonas maltophilia
Acinetobacter spp.
Many other
(H. influenza, etc..)
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DR.T.V.RAO MD 3
HISTORY OF RESISTANCE IN GRAM-NEGATIVE BACTERIA
1928
Fleming
1941
Penicillin use
1940 Penicillinase detected in
E. coli
1959
β -lactamase resistant penicillin's: Methicillin
1960s
Broad spectrum/ extended spectrum
penicillin's
1964
Cephalothin use
1965
Broad spectrum β –lactamases (TEM-1 in E. coli)
1983 Extended
spectrum β-lactamases
1950 1960 1970 1980 1990 2000
1985
Carbapenems (Imipenem)
Early 1980s
3rd generation ceph.
Carbapenemases
TEM-1 widespread
2005
Tigecycline
ESBL outbreaks in
France
1976
β –lactamases inhibitors
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4
DR.T.V.RAO MD
Plasmid-mediated TEM and SHV -lactamases
Ampicillin
1965
TEM-1E.coliS.paratyphi
1970s
TEM-1Reported in 28 Gm(-) sp
1983
ESBL in Europe
1988
ESBL in USA
2000
> 130 ESBLsWorldwide
Extended-spectrumCephalosporins
1963
Evolution of -Lactamases
Look and you will find ESBL
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DR.T.V.RAO MD 5
COMMITTEES CONTROLLING RESISTANCE PATTERNS
BSACUnited Kingdom
CA-SFMFrance
CLSI (NCCLS) USA
DINGermany
SRGASweden
CRGNetherlands
NWGANorway
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DR.T.V.RAO MD 6
STANDARDISATION• 1959 Ericsson & Steers – evaluation of methods
• 1961 WHO – standardisation
• 1964 Isenberg – comparison of methods in USA
• 1964 Truant – standardised tube dilution MICs
• 1966 Bauer-Kirby
• 1975 NCCLS CLSI• 1998 BSAC Standardised Method
• 2009 EUCAST Standardised Method
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DR.T.V.RAO MD 7
GROWING INCIDENCE OF ESBL PRODUCERS
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DR.T.V.RAO MD 8
Β-LACTAMASES CLASSIFICATION
• Molecular class:• A:
• TEM
• SHV
• other
• B: • Metalloenzymes
(carbapenemases)
• C: • Prototype: chromosomal
ampC
• D: • OXA (oxacillin
hydrolyzing enzymes)
• Enzyme type (by substrate profile):• Penicillinase
• Broad-spectrum
• Extended Spectrum
• Carbapenemase
• Genetic classification:• plasmids mediated
• Chromosomal
http://www.lahey.org/studies/webt.asp
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DR.T.V.RAO MD 9
TYPES OF Β-LACTAMASES
• β-lactamases
• Penicillinase: gene blaZ , inducible, on transposon (can move between chromosome and plasmid).
• Broad spectrum β-lactamases • (plasmid encoded)
• TEM
• SHV
• OXA (mainly in pseudomonas)
• ESBLs
• TEM related
• SHV related
• OXA related
• CTX-M
• Other
• ampC β-lactamases
• Resistant to β-lactamase inhibitors
• chromosomal
• Carbapenemases
• Metallo- β-lactamases
• Serine carbapenemases
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DR.T.V.RAO MD 10
EXTENDED-SPECTRUM -LACTAMASES ESBLS
• Mutations of TEM-1, TEM-2, SHV-1
• Inactivate -lactams with an oxyimino group (third-generation cephalosporins and aztreonam)
• Plasmid-mediated (often other R genes)
• 70 TEM and 15 SHV types
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DR.T.V.RAO MD 11
GENETIC MECHANISM
Transformation
Penicillinase blaZ
Plasmidtransfer
Broad spectrum b-lactamase
(blaTEM)
&
Mutation
ESBL(TEM related)
&
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DR.T.V.RAO MD 12
-LACTAMASE ACTIVITY
C C
C N
H H
R-CONH
S
COOH
CH3
CH3
O
Enzyme-Ser-OH
-lactam
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DR.T.V.RAO MD 13
-LACTAMASE ACTIVITY
C C
C N
H H
R-CONH
S
COOH
CH3
CH3
O
HO
Ser
Enzyme
HOH
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DR.T.V.RAO MD 14
L
L
L
LL
L
L
L Lb-lactamaseproduction
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DR.T.V.RAO MD 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
WHAT ARE ESBL PRODUCING BACTERIA.
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DR.T.V.RAO MD 16
ESBL• Confer resistance to 1st , 2nd, 3rd cephalosporins.
• Most are susceptible to β-lactamase inhibitors
• Most are susceptible to 4th cephalosporins
• All are susceptible to carbapenems
• Diversity of ESBL• SHV (widespread)
• TEM (>100 types)
• OXA • Predominantly in Pseudomonas
• less susceptible to β-lactamase inhibitors
• CTX-M• Probably independent evolution
• Highly resistant to 3rd generation cephalosporins
• initially in South America, Far East & Eastern Europe
• Probably most frequent worldwide
• Clonal spread has been documented
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FURTHER COMPLICATING MATTERS:
• More than one gene of β-lactamase / ESBL / ampC / carbapenemase can be carried on the same plasmid.
• Genes of ESBL are carried on plasmids that usually carry additional resistant genes: frequently MDR
• Laboratory diagnosis confusing:
susceptibility profiles sometimes
misleading: “hidden resistance” ->
CLSI guidelines are changing.
• CTX-M clones appearing in the
community (Canada, Greece, Spain,
Italy).DR.T.V.RAO MD 17
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DR.T.V.RAO MD 18
ESBLS INCLUDE:• Cephalosporin-hydrolysing mutants of TEM and SHV -
the common plasmid-mediated penicillinases of Enterobacteriaceae. Well over 100 such variants are known
• • 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.
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DR.T.V.RAO MD 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).
ESBL ARE IMPORTANT CAUSE OF RESISTANCE
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DR.T.V.RAO MD 20
THEY SHOULD BE DISTINGUISHEDFROM OTHER IMPORTANT MODES OF RESISTANCE TO 2ND
AND 3RD GENERATION CEPHALOSPORINS, EG:
• Hyper produced chromosomal AmpC β-lactamases, especially in Enterobacter species.
• • Plasmid-mediated AmpC β-lactamases, in Klebsiella spp. and E. coli (rare)
• • Hyper produced K1 chromosomal β-lactamases in K. oxytoca not pneumoniae)
• • Efflux-mediated resistance in P. aeruginosa
• • Various ill-defined mechanisms in Acinetobacter species.
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DR.T.V.RAO MD 21
LABORATORY DETECTION: SCREENING ANDCONFIRMATION
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DR.T.V.RAO MD 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.
PRIMARY TESTING FOR ESBL DETECTION
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DR.T.V.RAO MD 23
SCREENING• 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:
• TEM and SHV ESBLs – obvious resistance to ceftazidime, variable to cefotaxime
• CTX-M ESBLs – obvious resistance to cefotaxime: variable to ceftazidime
• All ESBLs – obvious resistance to cefpodoxime
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DR.T.V.RAO MD 24
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DR.T.V.RAO MD 25
DETECTION STRATEGY: STEP 1
See http://www.hpa.org.uk
• Screen Enterobacteriaceae with :• Cefpodoxime- best general ESBL substrate
• Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively
Spread of CTX-M into community means screening must be wider than before
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DR.T.V.RAO MD 26
DETECTION OF ESBLS: STEP 2
See http://www.hpa.org.uk
• Seek ceph/clav synergy in ceph R isolates
•Double disc•Combination disc•Etest
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DR.T.V.RAO MD 28
ESBL CONFIRMATORY TESTS Double-disk synergy (DDS) test
• CAZ and CAZ/CA disks• CTX and CTX\CA disks• Confirmatory testing requires using both CAZ and CTX alone and with CA
• 5 mm enhancement of the inhibition zone of antibiotic/CA combination vs antibiotic tested alone = ESBL
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DR.T.V.RAO MD 29
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DR.T.V.RAO MD 30
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DR.T.V.RAO MD 31
ESBL CONFIRMATION
Clavulanate Inhibition with
Cefotaxime - + +-
Ceftazidime - + - +
NOT ESBLreport results as they appear
ESBLreport cephalosporins, penicillins,
aztreonam as “R”
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DR.T.V.RAO MD 32
CTXCAZ
CTX+CA CAZ+CA
CMZTest for E. coli K. pneumoniaeK. oxytoca
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DR.T.V.RAO MD 33
ESBL Confirmatory TestPositive for ESBL
Ceftaz/CACefotax/CA
Ceftaz Cefotax
33
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DR.T.V.RAO MD 34
ESBL CONFIRMATORY TEST NEGATIVE FOR ESBL
Ceftaz/CA Cefotaxime/CA
Ceftaz Cefotax
34
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DR.T.V.RAO MD 35
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DR.T.V.RAO MD 36
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DR.T.V.RAO MD 37
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DR.T.V.RAO MD 38
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DR.T.V.RAO MD 39
ESBL EXAMPLEMIC (g/ml)
Cefotaxime >32
Ceftazidime 0.5
Cefotaxime/clavulanate 0.5
Ceftazidime/clavulanate 1.0
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DR.T.V.RAO MD 40
HOW TO REPORTKLEBSIELLA PNEUMONIAE (ESBL)
Ampicillin R
Amoxicillin/clavulanate S
Cefazolin R
Cefotiam R
Cefmetazole S
Cefotaxime I R
Aztreonam R
, NCCLS not recommended
Cefepime S (?) R
Gentamicin R
Amikacin S
Imipenem S
Ciprofloxacin S
Flomoxef S ?
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DR.T.V.RAO MD 41
• It follows that the logical indicator is either cefpodoxime or BOTH of cefotaxime and ceftazidime resistance.
• 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,
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DR.T.V.RAO MD 42
AMPC
• Gene on chromosome
• Produced by virtually all GNBs
• Activity generally low
• NOT inhibited by -lactamase inhibitors
• Differ in E. coli/Klebsiella versus other GNBs
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DR.T.V.RAO MD 43
AMPC -LACTAMASEE. COLI/KLEBSIELLA SPP.-TYPICAL
• Chromosomal
• Produced in minimal amount
• NOT inducible
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DR.T.V.RAO MD 44
AMPC -LACTAMASEE. COLI/KLEBSIELLA SPP.- GENETIC
CHANGES
• AmpC genes mutation
• Low amount-destroy ampicillin, 1st cephalosporins
• High amount-destroy expanded spectrum -lactams
• Transfer of ampC to plasmid
• Hyper production
• Resistant to 3rd cephalosporins, cephamycins
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DR.T.V.RAO MD 45
AMPC -LACTAMASEOTHER GNBS-TYPICAL
• Chromosomal
• Produced in small amount
• Inducible (hyper production)
• Reversible
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DR.T.V.RAO MD 46
Etest for ESBLs
Cefotaxime
Cefotaxime+
clavulanate
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DR.T.V.RAO MD 47
Etest for ESBLs
Cefotaxime
Cefotaxime+
clavulanate
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DR.T.V.RAO MD 48
QUALITY CONTROL IN ESBL DETECTION
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DR.T.V.RAO MD 49
• 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
CONTROLS FOR ESBL TESTS
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DR.T.V.RAO MD 50
• 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.
• 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.
CLSI RECOMMENDS
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DR.T.V.RAO MD 51
• 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.
ZONE DETERMINATION GUIDES …
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DR.T.V.RAO MD 52
• 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
ROLE OF CLSI IN REVISING BREAKPOINTS IN ANTIBIOTIC RESISTANCE
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DR.T.V.RAO MD 53
FOLLOW THE NEW GUIDELINES CLSI 2010
• 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.
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DR.T.V.RAO MD 54
WHY DO BREAKPOINTS SOMETIMES NEED
TO BE REVISED?
• 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.
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DR.T.V.RAO MD 55
AgentCLSI 2009 CLSI 2010
S I R S I R
Cefazolin ≤8 16 ≥32 ≤1 2 ≥4
Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4
Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4
Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16
Aztreonam ≤8 16 ≥32 ≤4 8 ≥16
Cefipime ≤8 16 ≥32 ≤8 16 ≥32
ENTEROBACTERIACEAE: REVISED BREAKPOINTS FOR CEPHALOSPORINS
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DR.T.V.RAO MD 56
• Agent Old (M100-S19) Revised (M100-S20)
S I R S I R
• Cefazolin ≥18 15-17 ≤14 NA NA NA
• Cefotaxime ≥23 15-22 ≤14 ≥26 23-25 ≤22
• Ceftizoxime ≥20 15-19 ≤14 ≥25 22-24 ≤21
• Ceftriaxone ≥21 14-20 ≤13 ≥23 20-22 ≤19
• Ceftazidime ≥18 15-17 ≤14 ≥21 18-20 ≤17
• Aztreonam ≥22 16-21 ≤15 ≥21 18-20 ≤17
• S – susceptible
• I – Intermediate
• R – Resistant.
DISK DIFFUSION BREAKPOINTS (MM):
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DR.T.V.RAO MD 57
• Agent M100-S19 M100-S20
S I R S I R
• Cefuroxime ≤8 16 ≥32 ≤8 16 ≥32
• Cefepime ≤8 16 ≥32 ≤8 16 ≥32
• Cefotetan ≤16 32 ≥64 ≤16 32 ≥64
• Cefoxitin ≤8 16 ≥32 ≤8 16 ≥32
• S – susceptible
• I – Intermediate
• R – Resistant
FOLLOWING MIC BREAKPOINTS WERE REEVALUATED FOR ENTEROBACTERIACEAE BUT WERE NOT REVISED
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DR.T.V.RAO MD 58
WHY WERE THE BREAKPOINTS FOR CEFEPIME AND CEFUROXIME (PARENTERAL) NOT REVISED?
• 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
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DR.T.V.RAO MD 59
WHY ARE THERE NO DISK DIFFUSION BREAKPOINTS FOR CEFAZOLIN?
• 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.
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DR.T.V.RAO MD 60
BACTERIA NOT TO TEST FOR ESBL’S
• Acinetobacter• Acinetobacter often S to clavulanate alone
• S. maltophilia• You get +ve results via inhibition of L-2
chromosomal b-lactamase, which is ubiquitous in the species
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DR.T.V.RAO MD 61
PITFALLS IN ESBL DETECTION
• 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
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DR.T.V.RAO MD 62
MOLECULAR DETECTION: WHERE AND WHY ?
• In the Reference Laboratory
– confirmation of unusual resistance– surveillance of resistance mechanisms– monitoring spread of resistance genes / strains– identify strains likely to contain novel resistance
mechanisms
• In the clinical diagnostic laboratory
– rapid detection for patient management– infection control
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DR.T.V.RAO MD 63
MOLECULAR DETECTION: DIFFERENT NEEDS
• In the Reference Laboratory,
– testing “pure” cultures– myriad assays and formats – numerous bug-drug combinations
• In the clinical diagnostic laboratory
– directly from specimens– need to target key species– format must be simple, rapid and cost-effective– problems with genes in commensals
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DR.T.V.RAO MD 64
• Simple and multiplex PCR
• Real-time PCR• DNA sequencing• Hybridisation-
based techniques
MOLECULAR DETECTION
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DR.T.V.RAO MD 65
MOLECULAR TESTS IN CLINICAL LABS
Black box approach:
molecular biology steps hidden
Simple end-product detection
Simple samplepreparation
• Must be rapid (TATs), inexpensive, reliable !• Platform must be sufficiently versatile to justify investment• Relatively hands-free, with scope for automation• On-going – e.g. <30 min test for ESBL detection
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Chips with everything…going beyond AST
!Total profiling; more cost-effective than PCR
• species identification• resistance genes• virulence genes• epidemicity predictors• strain-specific markers
DR.T.V.RAO MD 66
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DR.T.V.RAO MD 67
MOLECULAR DETECTION: THE INHERENT PROBLEM
• Molecular methods only detect known mechanisms• only as good as available sequence data• resistant isolates with known genes identified
• & new variants, if sufficient homology• false-resistance (unexpressed / partial genes)
• Susceptibility must always be confirmed• can’t base treatment on a negative molecular result • can’t detect genuinely new resistance mechanisms• will never (?) replace cheap phenotypic methods
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DR.T.V.RAO MD 68
HAND WASHING CAN REDUCE THE PREVALENCE AND SPREAD OF ESBL PRODUCERS
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DR.T.V.RAO MD 69
FOLLOW ME FOR ARTICLES OF INTEREST ON INFECTIOUS DISEASES AND MICROBIOLOGY ..
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DR.T.V.RAO MD 70
• Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical
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