EXTENDED SPECTRUM
B-LACTAMASES
(ESBL)
BASIS, DETECTION AND REPORTING
Dr.T.V.Rao MD
DR.T.V.RAO MD
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V. cholera
C. jejuni
Helicobacter pylori
Acinetobacter spp.
Gram Negative
Bacilli
Many other
(H. influenza, etc..)
Enterobacteriaceae
DR.T.V.RAO MD
Stenotrophomonas
maltophilia
Pseudomonas
aeruginosa
2
HISTORY OF RESISTANCE IN
GRAM-NEGATIVE BACTERIA
1965
1940
Penicillinase
detected in
E. coli
1950
1941
Broad spectrum β –
lactamases (TEM-1 in E. coli)
ESBL
outbreaks in
France
1983
Extended
TEM-1
widespread spectrum βlactamases
1960
1970
1964
1980
Carbapenemases
1990
2000
Early 1980s
1959
Cephalothin 3rd generation
Penicillin
use β -lactamase resistant use
ceph.
1985
penicillin's: Methicillin
1960s
Carbapenem
s (Imipenem)
Broad spectrum/
1976
extended
spectrum
1928
β –lactamases
penicillin's
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inhibitors
Fleming
2005
Tigecycline
3
Evolution of -Lactamases
Plasmid-mediated TEM and SHV -lactamases
Ampicillin
1963
1965
Extended-spectrum
Cephalosporins
1970s
1983
1988
2000
Look and you will find ESBL
D
R
.
T
.
V
.
R
TEM-1
TEM-1
E.coli
Reported in
S.paratyphi 28 Gm(-) sp
ESBL in
Europe
ESBL
in USA
> 130 ESBLs
Worldwide
4
COMMITTEES CONTROLLING
RESISTANCE PATTERNS
BSAC
SRGA
United Kingdom
Sweden
CA-SFM
CRG
France
Netherlands
DIN
NWGA
Germany
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Norway
CLSI (NCCLS)
USA
5
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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GROWING INCIDENCE OF ESBL
PRODUCERS
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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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TYPES OF Β-LACTAMASES
• β-lactamases
• Penicillinase: gene blaZ ,
inducible, on transposon (can
move between chromosome and
plasmid).
•
ESBLs
• TEM related
• SHV related
• OXA related
• CTX-M
• Other
• Broad spectrum β-lactamases
•
ampC β-lactamases
• Resistant to β-lactamase
inhibitors
• chromosomal
•
Carbapenemases
• Metallo- β-lactamases
• Serine carbapenemases
• (plasmid encoded)
• TEM
• SHV
• OXA (mainly in pseudomonas)
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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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GENETIC MECHANISM
Broad spectrum
b-lactamase
(blaTEM)
Penicillinase
blaZ
&
Transformation
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ESBL
(TEM related)
&
Plasmid
transfer
Mutation
11
-LACTAMASE ACTIVITY
H
H
S
R-CONH
C
C
C
N
-lactam
CH3
CH3
O
COOH
Enzyme-Ser-OH
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-LACTAMASE ACTIVITY
H
H
S
R-CONH
O
HOH
C
C
C
N
O
H
CH3
CH3
COOH
Ser
Enzyme
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L
L L
L




L





L
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L




 
L
-lactamase
production


L 



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WHAT ARE ESBL PRODUCING BACTERIA.
• 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
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ESBL
• Confer resistance to 1 st , 2nd, 3rd cephalosporins.
• Most are susceptible to β-lactamase inhibitors
• Most are susceptible to 4 th 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 3 rd 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).
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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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ESBL ARE IMPORTANT CAUSE OF
RESISTANCE
• ESBLs are not the sole βlactamases 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).
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THEY SHOULD BE DISTINGUISHED
FROM 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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LABORATORY DETECTION:
SCREENING AND
CONFIRMATION
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PRIMARY TESTING FOR ESBL DETECTION
• 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.
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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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DETECTION STRATEGY: STEP 1
• Screen Enterobacteriaceae with :
• Cefpodoxime- best general ESBL substrate
• Cefotaxime & ceftazidime- good substrates for CTXM & TEM/SHV, respectively
Spread of CTX-M into community means
screening must be wider than before
See http://www.hpa.org.uk
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DETECTION OF ESBLS: STEP 2
• Seek ceph/clav synergy in ceph R isolates
•Double disc
•Combination disc
•Etest
See http://www.hpa.org.uk
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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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ESBL CONFIRMATION
Clavulanate Inhibition with
Cefotaxime
-
Ceftazidime
-
-
+
+
+
-
+
ESBL
NOT ESBL
report results as they appear report cephalosporins, penicillins,
aztreonam as “R”
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CAZ+CA
CTX+CA
CMZ
CTX
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Test for
E. coli
K. pneumoniae
K. oxytoca
CAZ
32
ESBL Confirmatory Test
Positive for ESBL
Ceftaz/CA
Ceftaz
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Cefotax/CA
Cefotax
33
33
ESBL CONFIRMATORY TEST NEGATIVE FOR
ESBL
Ceftaz/CA
Ceftaz
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Cefotaxime/CA
Cefotax
34
34
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ESBL EXAMPLE
MIC (g/ml)
Cefotaxime
>32
Ceftazidime
0.5
Cefotaxime/clavulanate
0.5
Ceftazidime/clavulanate
1.0
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HOW TO REPORT
KLEBSIELLA PNEUMONIAE (ESBL)
Ampicillin
R
Cefepime
Amoxicillin/clavulanate
S
Gentamicin
Cefazolin
R
Cefotiam
R
Cefmetazole
S
Cefotaxime
I R
Aztreonam
R
, NCCLS
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S (?) R
R
Amikacin
S
Imipenem
S
Ciprofloxacin S
Flomoxef  S ?
not recommended
40
• 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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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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AMPC -LACTAMASE
E. COLI/KLEBSIELLA SPP.-TYPICAL
• Chromosomal
• Produced in minimal amount
• NOT inducible
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AMPC -LACTAMASE
E. COLI/KLEBSIELLA SPP.- GENETIC
CHANGES
• AmpC genes mutation
• Low amount-destroy ampicillin,
cephalosporins
• High amount-destroy expanded spectrum
1st
-lactams
• Transfer of ampC to plasmid
• Hyper production
• Resistant to 3rd cephalosporins, cephamycins
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AMPC -LACTAMASE
OTHER GNBS-TYPICAL
• Chromosomal
• Produced in small amount
• Inducible (hyper production)
• Reversible
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Etest for ESBLs
Cefotaxime
Cefotaxime
+
clavulanate
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Etest for ESBLs
Cefotaxime
Cefotaxime
+
clavulanate
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QUALITY CONTROL IN
ESBL DETECTION
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CONTROLS FOR ESBL TESTS
• 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 (broadspectrum) NCTC 13351
TEM-10 (ceftazidimase)
NCTC 13352
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CLSI RECOMMENDS
•
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.
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ZONE DETERMINATION GUIDES …
• 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.
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ROLE OF CLSI IN REVISING BREAKPOINTS IN
ANTIBIOTIC RESISTANCE
• 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
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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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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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ENTEROBACTERIACEAE: REVISED
BREAKPOINTS FOR CEPHALOSPORINS
CLSI 2009
Agent
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
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DISK DIFFUSION BREAKPOINTS (MM):
• Agent
Old (M100-S19)
S
I
Revised (M100-S20)
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.
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FOLLOWING MIC BREAKPOINTS WERE REEVALUATED FOR
ENTEROBACTERIACEAE BUT WERE NOT REVISED
• 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
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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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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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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 -lactamase, which is ubiquitous
in the species
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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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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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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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MOLECULAR DETECTION
• Simple and
multiplex PCR
• Real-time PCR
• DNA sequencing
• Hybridisationbased techniques
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MOLECULAR TESTS IN CLINICAL LABS
Simple sample
preparation
Black box
approach:
molecular biology
steps hidden
Simple end-product
detection
• 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
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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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HAND WASHING CAN REDUCE THE PREVALENCE
AND SPREAD OF ESBL PRODUCERS
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FOLLOW ME FOR ARTICLES OF INTEREST ON
INFECTIOUS DISEASES AND MICROBIOLOGY ..
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• Created by Dr.T.V.Rao MD for
‘ e ‘ learning resources for Medical
Professionals in the Development World
• Email
• doctortvrao@gmail.com
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