HODGE TEST

Report
HODGE TEST
SKILL BASED LEARNING
Dr.T.V.Rao MD
Dr.T.V.Rao MD
1
Role Carbapenems
• Carbapenems are often used as antibiotics of
last resort for treating infections due to
multidrug-resistant gram-negative bacilli,
because they are stable even in response to
extended spectrum and AmpC -lactamases.
However, gram-negative bacilli producing the
acquired metallo-lactamases (MBLs) IMP and
VIM have been increasingly reported in Asia
and Europe
Dr.T.V.Rao MD
2
Emerging Carbapenems Resistance in
Gram-Negative Bacilli
• Significantly limits treatment options for lifethreatening infections
• No new drugs for gram-negative bacilli
• Emerging resistance mechanisms,
carbapenemases are mobile,
• Detection of carbapenemases and
implementation of infection control practices
are necessary to limit spread
Dr.T.V.Rao MD
3
Antibiotic Misuse
• Antibiotic misuse, (sometimes called antibiotic
abuse or antibiotic overuse) refers to the misuse
and overuse of antibiotics which has serious
effects on public health. Antibiotic resistant
bacteria is a growing threat and becoming
increasingly common. This overuse creates multiantibiotic resistant life threatening infections by
"super bugs”, sometimes out of relatively
harmless bacteria. Antibiotic abuse also places
the patient at unnecessary risk of adverse effects
of antibiotics.
Dr.T.V.Rao MD
4
Bugs to Superbugs.
• Antibiotic resistance
develops through gene
action or plasmid
exchange between
bacteria of the same
species. If a bacterium
carries several
resistance genes, it is
called multiresistant or,
informally, a superbug.
Dr.T.V.Rao MD
5
Carbapenem Resistance:
Mechanisms
Enterobacteriaceae
Cephalosporinase + porin loss
Carbapenemase
P. aeruginosa
Porin loss
Up-regulated efflux
Carbapenemase
Acinetobacter spp.
Cephalosporinase + porin loss
Carbapenemase
Carbapenemases
Classification
Enzyme
Most Common Bacteria
Class A
KPC, SME,
IMI, NMC,
GES
Enterobacteriaceae
(rare reports in P. aeruginosa)
Class B
IMP, VIM,
(metallo-b-lactamse) GIM, SPM
P. aeruginosa
Enterobacteriacea
Acinetobacter spp.
Class D
Acinetobacter spp.
OXA
Klebsiella Pneumoniae
Carbapenemase
• KPC is a class A b-lactamase
– Confers resistance to all b-lactams including extended-spectrum
cephalosporins and carbapenems
• Occurs in Enterobacteriaceae
– Most commonly in Klebsiella pneumoniae
– Also reported in: K. oxytoca, Citrobacter freundii, Enterobacter
spp., Escherichia coli, Salmonella spp., Serratia spp.,
• Also reported in Pseudomonas aeruginosa (Columbia)
Dr.T.V.Rao MD
8
KPC’s in Enterobacteriaceae
Species
Comments
Klebsiella spp.
K. pneumoniae-cause of outbreaks
K. oxytoca-sporadic occurrence
Enterobacter spp.
Escherichia coli
Salmonella spp.
Sporadic occurrence
Citrobacter freundii
Serratia spp.
Pseudomonas aeruginosa – Columbia & Puerto Rico
What Labs Should Do Now
• Look for isolates of Enterobacteriaceae
(especially K. pneumoniae), with carbapenem
MIC ≥ 2 mg/ml or non susceptible to ertapenem
by disk diffusion
• Consider confirmation by Modified Hodge Test
• Alert clinician and infection control practitioner
to possibility of mobile carbapenemase in
isolate
Dr.T.V.Rao MD
10
Control of Infections With Carbapenem-Resistant or
Carbapenemase-Producing Enterobacteriaceae in
Acute Care Facilities
• A difficulty in detecting CRE is the fact that
some strains that harbor blakpc have minimal
inhibitory concentrations (MICs) that are
elevated but still within the susceptible range
for carbapenems. Because these strains are
susceptible to carbapenems, they are not
identified as potential clinical or infection
control risks using current susceptibility
testing guidelines.
Dr.T.V.Rao MD
11
Background to Modified Hodge
Test
• KPCs are class A carbapenemases that reside on
transferable plasmids and are capable of inactivating
carbapenems, such as imipenem and meropenem.
Since carbapenems are often used to treat infections
caused by extended-spectrum beta lactamase (ESBL)producing Gram-negative bacteria, carbapenemase
production in Enterobacteriaceae can significantly
limit treatment options for life-threatening diseases.
KPCs occur most commonly in Klebsiella pneumoniae
but have been seen in other species of
Enterobacteriaceae as well.
Dr.T.V.Rao MD
12
CLSI – Modified Hodge Test
• CLSI published a
recommendation that
carbapenems-susceptible
Enterobacteriaceae
with elevated MICs or
reduced disk diffusion
zone sizes be tested for
the presence of
carbapenemases using
the modified Hodge test
(MHT).
Dr.T.V.Rao MD
13
Modified Hodge Test
• The Modified Hodge Test (MHT) detects
carbapenemase production in isolates of
Enterobacteriaceae. The most common
carbapenemase found in Enterobacteriaceae is
the Klebsiella pneumoniae carbapenemase (KPC).
Other carbapenemase, like the metallo β
lactamase (MBL) and the SME-1 in Serratia
marcescens, can also produce a positive MHT, but
are found infrequently in the United States.
Dr.T.V.Rao MD
14
Purpose of Hodge Test
• Carbapenemase
production is detected by
the MHT when the test
isolate produces the
enzyme and allows
growth of a carbapenems
susceptible strain (E.coli
ATCC 25922) towards a
carbapenem disk. The
result is a characteristic
cloverleaf-like indentation
Dr.T.V.Rao MD
15
Modified Hodge Test
• The MHT performed on
a 100 mm MHA plate.
(1) K. pneumoniae ATCC
BAA 1705, positive
result (2) K.
pneumoniaeATCC BAA
1706, negative result;
and (3) a clinical isolate,
positive result312
Dr.T.V.Rao MD
16
Reagents and materials
Reagents
•
•
•
•
•
•
•
•
•
•
•
5 ml Mueller Hinton broth (MHB) or 0.85% physiological saline
Mueller Hinton agar (MHA)
10 μg meropenem or ertapenem susceptibility disk
E. coli ATCC 25922: 18–24hr subculture
Equipment
Turbidity meter
35OC ± 2OC ambiant air incubator
Supplies
Sterile cotton-tipped swabs
1 ml sterile pipette
Sterile loop
Dr.T.V.Rao MD
17
Specimen and Quality Control
•
•
•
•
•
•
Specimen
Test organisms: 18–24 hr subculture
Special safety precautions
Biosaftey Level 2
Quality control
Perform quality control of the carbapenem disks
according to CLSI guidelines.
• Perform quality control with each run.
• MHT Positive Klebsiella pneumoniae ATCC BAA-1705
• MHT Negative Klebsiella pneumoniae ATCC BAA-1706
Dr.T.V.Rao MD
18
Step 1 and 2
• 1.Prepare a 0.5
McFarland dilution of
the E.coli ATCC 25922 in
5 ml of broth or saline.
• 2Dilute 1:10 by adding
0.5 ml of the 0.5
McFarland to 4.5 ml of
MHB or saline
•
Dr.T.V.Rao MD
19
Step 3 and 4
• Streak a lawn of the 1:10
dilution of E.coli ATCC
25922 to a Mueller
Hinton agar plate and
allow to dry 3–5 minutes.
• Place a 10 μg meropenem
or ertapenem
susceptibility disk in the
center of the test area.
•
Dr.T.V.Rao MD
20
Step 5 and 6
• In a straight line, streak
test organism from the
edge of the disk to the
edge of the plate. Up to
four organisms can be
tested on the same
plate with one drug.
• Incubate overnight at
35OC ± 2OC in ambient
air for 16–24 hour
•
Dr.T.V.Rao MD
21
Interpretation of MHT
• After 16–24 hours of
incubation, examine the
plate for a clover leaftype indentation at the
intersection of the test
organism and the E. coli
25922, within the zone
of inhibition of the
carbapenem
susceptibility disk.
Dr.T.V.Rao MD
22
MHT – Positive Test
• A positive MHT indicates
that this isolate is
producing a
carbapenemase
• Test has a clover leaflike indentation of the
E.coli 25922 growing
along the test
organism growth
streak within the disk
diffusion zone.
Dr.T.V.Rao MD
23
Showing the Results on HMT
testing
• A positive test indicates
carbapenemase production
by the test microorganism.
By producing
carbapenemase, the test
microorganism is able to
inactivate the carbapenem
that diffuses from the disk
after the disk has been
placed on the Mueller
Hinton Agar. This allows
carbapenem susceptible E.
coli ATCC® 25922™* to grow
toward the disk
Dr.T.V.Rao MD
24
MHT – Negative Test
• A negative MHT
indicates that this
isolate is not producing
a carbapenemase
• Test has no growth of
the E.coli 25922 along
the test organism
growth streak within
the disc diffusion.
Dr.T.V.Rao MD
25
Showing Positive and Negative
isolates by HMT
• Isolates A and C are
negative for KPC.
Isolates B and D are
positive for KPC as
indicated by arcing
growth of the
carbapenem-sensitive E
coli along the clinical
KPC isolates toward the
carbapenem disk.
Dr.T.V.Rao MD
26
What Acquired metallo—lactamases do
(MBL)
• The MBLs efficiently
hydrolyze all -lactams,
except for aztreonam,
in vitro . Therefore,
detection of MBLproducing gramnegative bacilli is crucial
for the optimal
treatment of patients
and to control the
spread of resistance
Dr.T.V.Rao MD
27
Lee et al - reports Carbapenemase
detection methods
• Lee et al. have reported
that the Hodge test can
be used to screen
carbapenemaseproducing gram-negative
bacilli and that the
imipenem (IPM)-EDTA
double-disk synergy test
(DDST) can distinguish
MBL-producing from
MBL-nonproducing gramnegative bacilli
Dr.T.V.Rao MD
28
Phenotypic detection with Hodge test a
Minimal requirement
• Carbapenem resistance
and carbapenemase
production conferred by
blaNDM-1 is detected
reliably with phenotypic
testing methods currently
recommended by the
Clinical and Laboratory
Standards Institute ,
including disk diffusion
testing and the modified
Hodge test
Dr.T.V.Rao MD
29
Modified Hodge Test
Lawn of E. coli ATCC 25922
1:10 dilution of a
0.5 McFarland suspension
Test isolates
Imipenem disk
Dr.T.V.Rao
Described by Lee et al. CMI, 7, 88-102.
2001.MD
30
Modified Hodge Test
• Preliminary results suggest that any of the three
carbapenem disks work in the Modified Hodge
Test
Dr.T.V.Rao MD
31
What Labs Should Do Now
• Look for isolates of Enterobacteriaceae (especially
K. pneumoniae), with carbapenem MIC ≥ 2 mg/ml
or non susceptible to ertapenem by disk diffusion
• Consider confirmation by Modified Hodge Test
• Can submit initial isolate to CDC via NJ State Lab
for confirmation by blaKPC PCR if KPC-producers not
previously identified in hospital’s isolate
population
• Alert clinician and infection control practitioner to
possibility of mobile carbapenemase in isolate
Dr.T.V.Rao MD
32
Are we losing the Value of
Carbapenems
• Carbapenems are the only
antibiotics reliably active
against many otherwise
multi-resistant gramnegative opportunist
bacteria, particularly those
with extended-spectrum
beta-lactamases (ESBLs) The
growing emergence and
diversity of carbapenemase
producing strains is
therefore a major concern.
Dr.T.V.Rao MD
33
CDC reports the new genetic
mechanisms
• The isolate, Klebseilla pneumoniae 05-506, was
shown to possess a metallo-beta-lactamase
(MBL) but was negative for previously known
MBL genes. Gene libraries and amplification of
class 1 integrons revealed three resistanceconferring regions; the first contained bla(CMY-4) flanked
by ISEcP1 and blc. The second region of 4.8 kb
contained a complex class 1 integron with the
gene cassettes arr-2, a new erythromycin
esterase gene; ereC; aadA1; and cmlA7
Dr.T.V.Rao MD
34
How we can Improve Hodge Test
• The Hodge test is a simple method for
screening MBL-producing isolates, but
occasional isolates show false-negative
results. The test can be improved by using an
IPM disk to which 10 l of 50 mM zinc sulfate
(140 g/disk) has been added (Table 3) or by
using Mueller-Hinton agar to which zinc
sulfate has been added to a final
concentration of 70 g/m
Dr.T.V.Rao MD
35
Genetic origin of the NDM-1
• An intact ISCR1 element was shown to be downstream
from the qac/sul genes. The third region consisted of a
new MBL gene, designated bla(NDM-1), flanked on one
side by K. pneumoniae DNA and a truncated IS26
element on its other side. The last two regions lie
adjacent to one another, and all three regions are
found on a 180-kb region that is easily transferable to
recipient strains and that confers resistance to all
antibiotics except fluoroquinolones and colistin. NDM1 shares very little identity with other MBLs, with the
most similar MBLs being VIM-1/VIM-2, with which it
has only 32.4% identity.
Dr.T.V.Rao MD
36
Molecular configuration of NDM-1
• NDM-1 also has an additional insert
between positions 162 and 166 not
present in other MBLs. NDM-1 has a
molecular mass of 28 kDa, is monomeric,
and can hydrolyze all beta-lactams except
aztreonam. Compared to VIM-2, NDM-1
displays tighter binding to most
Cephalosporins.
Dr.T.V.Rao MD
37
NDM genetic coding differs from
other recent isolates
• Compared to VIM-2, NDM-1 displays tighter
binding to most cephalosporins, in particular,
cefuroxime, cefotaxime, and cephalothin
(cefalotin), and also to the penicillins. NDM-1
does not bind to the carbapenems as tightly as
IMP-1 or VIM-2 and turns over the carbapenems
at a rate similar to that of VIM-2. In addition to K.
pneumoniae 05-506, bla(NDM-1) was found on a
140-kb plasmid in an Escherichia coli strain
isolated from the patient's feces, inferring the
possibility of in vivo conjugation
Dr.T.V.Rao MD
38
Phenotypic detection with Hodge test a
Minimal requirement
• Carbapenem resistance
and carbapenemase
production conferred by
blaNDM-1 is detected
reliably with phenotypic
testing methods currently
recommended by the
Clinical and Laboratory
Standards Institute ,
including disk diffusion
testing and the modified
Hodge test
Dr.T.V.Rao MD
39
Follow me for More Articles of Interest on
Infectious Diseases
Dr.T.V.Rao MD
40
Created by Dr.T.V.Rao MD for
“ e” learning resources for Medical
Microbiologists in Developing
Countries
Email
[email protected]
Dr.T.V.Rao MD
41

similar documents