Claves para Identificar Microorganismos de Interes Clinico

OUTLINE
Overview of CLSI Document M35-A2

I. Introduction of concept
For Bench-level Identification of
Clinically-significant Microorganisms II. Major players
III. Application one
Erik Munson A. CLSI M35-A2
B. Perhaps why you are here
Clinical Microbiology
Wheaton Franciscan Laboratory IV. Application two
Milwaukee, Wisconsin A. Routine (but possibly covert) bench dealings
B. Help out WSLH
The presenter states no conflict of interest and has no financial relationship
to disclose relevant to the content of this presentation.
1 2
INTRODUCTION TO CLSI M35-A2

Goes way back to the introduction of commercial
identification systems
Laboratories lacking confidence, resources in
Introduction validation of alternative methods
Utilization of these methods has resulted in

greater standardization and more accuracy
In some cases, may have resulted in cost and

turnaround time increases
3 4
INTRODUCTION TO CLSI M35-A2 BENEFITS OF RAPID RESULTS

Laboratorians have used rapidly-determined
characteristics for years; this document seeks Turnaround Length of Mortality Average
Study Notes
to standardize this Time P Stay P 4 Index P 4 Cost P 4
Doern et al.1 < 0.0005 NS5 < 0.02 0.01 Rapid MicroScan product
Odor Barenfanger et al. 2 0.0014 0.006 0.45 0.04 VITEK product
Immediate enzymatic al 3
Kerremans et al. <0
0.0001
0001 ND6 0 21
0.21 ND VITEK products
reactions (spot testing)

Cost savings associated with using rapid methods 1J.
Clin. Microbiol. 32: 1757-1762; 1994
2J.
or overall patient care benefits Clin. Microbiol. 37: 1415-1418; 1999
3J. Antimicrob. Chemother. 61: 428-435; 2008
Chromogenic medium Slide Fluorogenic 4Role of rapid susceptibility testing also factored into this calculation
Agglutination Spot Enzymatic 5Not significant
Macroscopic, microscopic Disk Single-tube 6Not determined
5 6
1
WARNING CAVEATS
Laboratory directors, managers, and Isolates conforming to reactions described in
supervisors responsible for ensuring M35-A2 identify organism with >95% accuracy;
appropriate use of rapid methods identification can be reported without qualification
“Isolates to be tested should match “Confirmation by additional procedures is
th criteria
the it i required
i d ffor proper unnecessary for
f many off the
th species
i described
d ib d
identification.” in this document.”
CLSI M35-A2; 2008 CLSI M35-A2; 2008
Competency assessment Lack of a positive result does not rule out the
identification of an isolate; just signifies need for
Colony, Gram stain
Smell (when safe) additional testing
7 8
MORE WARNINGS EXCLUSIONS????

Employ standard precautions Rapid results may need to be validated
Cover transmission of all infectious agents Certain microbes from normally-sterile sites
Universal precautions cover blood-borne pathogens Potential agents of bioterrorism
Refer to CLSI M29 Microbes important to infection control practitioners
Microbes implicated
p in nosocomial outbreaks
Sniffing/wafting can be dangerous
Once mold colony is ruled out, opening Risk (being wrong) vs. benefit (patient care/safety)
of plates from non-invasive sources (i.e.,
urine, sputum) is common & relatively safe M35-A eliminated certain organism groupings
P. aeruginosa, H. influenzae, Eikenella, However, possibility of serious pathogen or ability

S. anginosus group odor can be detected to rule out potential agent of bioterrorism has
by opened plates “not sniffed purposely” resulted in M35-A2 including additional organisms
9 10
CLSI M35-A2 PROTOCOLS

Test descriptions
Principle Interpretation
Reagents Limitations (precautions)
Procedure Quality control
Major Players Thirteen reagents/tests

g
Bile solubility Indoxyl acetate
Rapid spot CAMP MUG test
Catalase (aerobes) Spot oxidase
Catalase (anaerobes) -aminolevulinic acid
Germ tube Rapid trehalose
Rapid hippurate hydrolysis Urea/phenylalanine
Spot indole deaminase
11 12
2
CATALASE SPOT INDOLE
3% hydrogen peroxide 5% (w/v) p-dimethylaminobenzaldehyde
1% paradimethylaminocinnamaldehyde in HCl
Touch center of isolated colony with stick
Moisten piece of filter paper with reagent
Transfer to clean glass slide
Place drop of hydrogen peroxide onto cell paste Rub portion of CFU from blood agar onto paper
G
Growth
th medium
di mustt contain
t i sufficient
ffi i t tryptophan
t t h
“Immediate” bubbling (< 20 seconds) is positive
Pigment formation (< 20 seconds) is positive
No non-platinum loops; don’t grab blood agar Cannot use media containing dyes
If slow-growing isolate on blood agar (no growth Cannot use Mueller-Hinton or high-glucose agar
MacConkey) or small GNR, perform test in Detectable indole diffuses to adjacent colonies
biological safety cabinet within 5 mm (false-positive results)
13 14
SPOT OXIDASE UREASE TUBE

1% tetramethyl-p-phenylenediamine dihydrochloride
20 g/L urea
Moisten piece of filter paper with reagent
Rub portion of CFU from blood agar onto paper Inoculate with large loopful of growth;
Use wooden stick or p
platinum bacteriological
g loop
p Incubate 35°C ambient air for two hours
Blue/purple pigment (< 10 seconds) is positive Deep pink color of broth indicates urease-positive
Cannot use MacConkey or other purple agar organisms; timing of reaction is important
Nickel-based alloy wires containing chromium
or iron may yield false-positive results upon Positive reactions can be followed up with by
organism transfer phenylalanine deaminase testing
15 16
Brucella spp.
Presumptive identification
Tiny Gram-negative coccobacillus Oxidase-positive
No growth on MacConkey Catalase-positive
Additional tests for definitive identification
M35 A2 Rapid Identification
M35-A2 Urease-positive (quick with disk) Indole-negative
Non-hemolytic on blood agar
Notes
Highly infectious; work in biological safety cabinet
Sterile tissues and fluids
Refer to Laboratory Response Network laboratory
17 18
3
Brucella spp. Campylobacter jejuni/coli
Presumptive identification Presumptive identification
Tiny Gram-negative coccobacillus Oxidase-positive Gram-negative bacilli (gull wing) Oxidase-positive
No growth on MacConkey Catalase-positive Darting motility Catalase-positive
Additional tests for definitive identification Additional tests for definitive identification
Urease-positive (quick with disk) Indole-negative Hippurate-positive Campylobacter jejuni
Non-hemolytic on blood agar Indoxyl acetate-positive Campylobacter jejuni/coli
Notes LIMITATIONS/additional factors Notes
Highly infectious;
Oligella ureolytica, work in biological
Bordetella bronchiseptica, safety cabinet
and Haemophilus influenzae Isolated colonies on Campylobacter-selective
Sterile tissues and fluids
can resemble Brucella spp. medium incubated in microaerophilic 42°C
Refer to spp.
Brucella Laboratory Response
are phenylalanine Network laboratory
deaminase-negative
19 20
Cardiobacterium hominis Cardiobacterium hominis

Pleomorphic, thin GNR Oxidase-positive Pleomorphic, thin GNR Oxidase-positive
No growth on MacConkey Catalase-negative No growth on MacConkey Catalase-negative
Indole-positive Indole-positive
Non-hemolytic on blood agar; may pit Non-hemolytic on blood agar; may pit
Notes Notes LIMITATIONS/additional factors
Isolate must be derived from blood culture Isolate
Pasteurella bettyaemust be derived
has same from blood
rapid biochemical profile culture
as C. hominis, but is
Confirm with negative nitrate test Confirm with negative
not known nitrate
to cause test
endocarditis
Rosette-forming, thin GNR (with above biochemicals) in adult blood cultures
21 22
Eikenella corrodens Escherichia coli

Small GNR; pits blood, chocolate Oxidase-positive Gram-negative bacilli via Gram Oxidase-negative
No growth on MacConkey Catalase-negative or growth on selective medium Indole-positive
Indole-negative Distinct odor of bleach Hemolytic OR lactose-positive AND PYR-negative
Non-hemolytic on blood agar OR lactose-negative AND MUG-positive
Notes Notes
Capnophilic Isolate must be growing as large colonies
The only MacConkey-negative, catalase-negative, Isolate cannot be derived from gastrointestinal source
oxidase-positive GNR that is ornithine-positive
23 24
4
Escherichia coli Escherichia coli
Gram-negative bacilli via Gram Oxidase-negative
or growth on selective medium Indole-positive
Hemolytic OR lactose-positive AND PYR-negative
OR lactose-negative AND MUG-positive
Notes LIMITATIONS/additional factors
Isolate
Occasional mustspp.
Shigella be can
growing as large colonies
be indole-positive and MUG-positive;
Isolate
therefore, cannot
no rapid be derived
identification of E. colifrom gastrointestinal
from blood, source
fecal, GI sources
CLSI M35-A2; 2008 Positive MUG test may be preliminary rule-out test for O157:H7
25 26
Francisella tularensis Haemophilus influenzae

Tiny GNR or coccobacillus (sand) Oxidase-negative Gram-negative coccobacillus
Growth on chocolate (48-72h) Catalase-neg/wk Good growth on chocolate; not blood, MacConkey
-lactamase-positive -aminolevulinic acid-negative
Notes Notes
Highly infectious; work in biological safety cabinet Satellitism on blood agar around Staphylococcus
No satellitism on blood agar around Staph streak streak separates Haemophilus from Brucella
Refer to Laboratory Response Network laboratory and Francisella
27 28
Haemophilus influenzae Kingella kingae

Gram-negative coccobacillus Gram-negative coccoid bacillus Oxidase-positive
Good growth on chocolate; not blood, MacConkey No growth on MacConkey Catalase-negative
-aminolevulinic acid-negative Hemolytic colonies on blood agar

Rapid Satellitism on blood
identification algorithm agartoaround
applies CSF andStaphylococcus
respiratory isolates Joint fluids, blood cultures, other sterile body sites
streak separates Haemophilus from Brucella Early growth confused for -hemolytic streptococci
Haemophilus haemolyticus cannot be differentiated from H. influenzae, with
and Francisella
exception of hemolysis characterization on horse blood agar
29 30
5
Moraxella catarrhalis Moraxella catarrhalis
Gram-negative diplococci Oxidase-positive Gram-negative diplococci Oxidase-positive
Hockey puck on blood agar Catalase-positive Hockey puck on blood agar Catalase-positive
Butyrate esterase-positive Butyrate esterase-positive
Indoxyl acetate-positive Indoxyl acetate-positive
Broth microdilution and disk diffusion susceptibility Broth
Most other microdilution
Moraxella and disk
spp. are positive diffusion
for butyrate susceptibility
esterase, but are
testing guidelines described in M45-A2 testing guidelines
coccobacilli described in M45-A2
rather than diplococci
31 32
Neisseria meningitidis Neisseria lactamica

Gram-negative diplococci Oxidase-positive Gram-negative diplococci Oxidase-positive
Glistening non-hemolytic growth on blood agar Graying non-hemolytic growth on blood agar
-glutamyl-aminopeptidase-positive -galactosidase-positive
Notes Notes
Highly infectious; work in biological safety cabinet
33 34
Neisseria gonorrhoeae Proteus mirabilis

Gram-negative diplococci Oxidase-positive Spreading colony Indole-negative
Strongly catalase-positive with 30% H2O2
-galactosidase-negative None, if susceptible to ampicillin
-gluatmyl-aminopeptidase-negative
Notes Notes (if resistant to ampicillin)
Growth on N. gonorrhoeae-selective medium Proteus spp. Ornithine decarboxylase Maltose fermentation
No growth on Mueller-Hinton or tryptic soy media P. mirabilis positive negative
P. penneri negative positive
35 36
6
Proteus mirabilis Proteus vulgaris
Spreading colony Indole-negative Spreading colony Indole-positive
None, if susceptible to ampicillin
Notes (if resistant to ampicillin) factors

LIMITATIONS/additional Notes
Proteus
Instead spp. maltose
of pursuing Ornithine decarboxylase
or ornithine Maltose
decarboxylase fermentation
testing, one could
report ampicillin-resistant
P. mirabilis (indole-negative) Proteae
positive as
negative
“indole-negative Proteus”
P. penneri negativeor P. mirabilis/P. penneri
positive
37 38
Pseudomonas aeruginosa Pseudomonas aeruginosa

Indole-negative Oxidase-positive Indole-negative Oxidase-positive
Metallic/pearlescent, rough, pigmented, mucoid Metallic/pearlescent, rough, pigmented, mucoid
Grape-like odor; corn tortilla Grape-like odor; corn tortilla

Most often strongly -hemolytic on blood agar Most
Rare often spp.
Aeromonas strongly beta-hemolytic
may resemble on blood
P. aeruginosa, but can agar
be
differentiated by positive spot indole test
Burkholderia cepacia isolates from CF patients can resemble P. aeruginosa
39 40
Staphylococcus aureus Staphylococcus aureus

Catalase-positive Catalase-positive
Tube/slide coagulase- or latex agglutination-positive Tube/slide coagulase- or latex agglutination-positive
Typically -hemolytic colonies on blood agar Typically -hemolytic colonies on blood agar

Tube coagulase required if non-hemolytic isolates Tube
S. schleiferi andcoagulase required
S. lugdunensis if non-hemolytic
may be slide coagulase-positive (clumpy,
from urine rather than complete agglutination); aforementioned species PYR-positive
S. saprophyticus may yield positive latex agglutination results
41 42
7
Staphylococcus lugdunensis Enterococcus spp.
Gram-positive cocci Catalase-positive GPC prs, chains (no clusters) Catalase-negative
Tube coagulase-negative Non--hemolytic on blood agar (>1 mm diameter)
PYR-positive (deep ruby red); PYR-positive
Polymyxin B-resistant; ornithine-positive
Notes Notes
May be slide coagulase-positive or clumpy Demonstrate an LAP-positive reaction with
-hemolytic Enterococcus spp. (improved specificity)
43 44
Enterococcus spp. Aerococcus viridans

GPC prs, chains (no clusters) Catalase-negative GPC (tetrads, clusters) Catalase-negative
Non--hemolytic on blood agar (>1 mm diameter) -hemolytic
PYR-positive PYR-positive
LAP-negative
Demonstrate
Lactococcus garvieae an
mayLAP-positive
be misidentifiedreaction with spp.
as Enterococcus
-hemolytic Enterococcus spp. (improved specificity)
45 46
Listeria monocytogenes Listeria monocytogenes

Small Gram-positive bacillus Catalase-positive Small Gram-positive bacillus Catalase-positive
Usually tumbling motility Small -hemolysis Usually tumbling motility Small -hemolysis
Hippurate-positive Hippurate-positive

Isolate should be from blood culture or CSF culture Isolate
Presence of should
catalase is not abe fromfactor
required blood forculture or CSF
identification in an culture
otherwise
Listeria can be non-motile after 35-37°C incubation Listeria can be non-motile
typical isolateafter 35-37°C incubation
Species confirmation not necessary; 95% of clinical isolates monocytogenes
47 48
8
Streptococcus agalactiae Streptococcus agalactiae
GPC (pairs, chains) Catalase-negative GPC (pairs, chains) Catalase-negative
Small zone of -hemolysis on blood agar Small zone of -hemolysis on blood agar
Hippurate-positive OR CAMP-positive Hippurate-positive OR CAMP-positive
OR Lancefield group B via latex agglutination OR Lancefield group B via latex agglutination
Hippurate method not to be used on non-hemolytic HippurateEnterococcus
-hemolytic method not tocan
spp. be be
used on non-hemolytic
hippurate-positive
colonies colonies
Many viridans group streptococci and non-hemolytic S. agalactiae are
hippurate-positive
49 50
Streptococcus anginosus group Streptococcus pneumoniae

GPC (pairs, chains) Catalase-negative GPC (lancet-shaped in pairs) Catalase-negative
CFU (<0.5 mm diameter); variable hemolysis -hemolysis on blood agar
Odor of butterscotch or vanilla OR Bile solubility-positive
Lancefield group F by latex agglutination
Notes Notes
May be Lancefield group A, C, F, or G by latex ~1% of S. pneumoniae with typical colony
agglutination morphology may not be bile soluble
51 52
Streptococcus pyogenes Viridans group Streptococcus

GPC (pairs, chains) Catalase-negative GPC (pairs, chains) Catalase-negative
CFU (>0.5 mm diameter); sharp -hemolysis Non-hemolytic or -hemolysis
PYR-positive OR PYR-positive LAP-positive
Lancefield group A by latex agglutination Bile solubility-negative if -hemolytic
Notes Notes
Careful observation of size and hemolysis, as Aerococcus urinae are cocci in clusters/tetrads
Enterococcus spp. can demonstrate -hemolysis Pediococcus spp. are cocci in clusters/tetrads
53 54
9
Candida albicans Candida glabrata
Budding yeast Small yeast in smear with no hyphae
Better growth on chocolate agar than blood agar
“Feet” in less than 48 hours OR Better growth on EMB agar than blood agar
Germ tube-positive Rapid trehalose-positive at 42°C
Notes Notes
Not easily separated from C. dubliniensis
55 56
Cryptococcus neoformans ANAEROBIC GRAM-NEGATIVES

Spherical pleomorphic budding yeast with no hyphae

Urease-positive
Phenol oxidase-positive
Notes
Cannot differentiate from C. gattii
57 CLSI M35-A2; 2008 58
ANAEROBIC GRAM-POSITIVES
A Second Application
CLSI M35-A2; 2008 59 60
10
CLSI M35-A2 AND BIOTERRORISM MORE…
Changes in recommendations for handling cultures
on the basis of unsuspected exposure to agents Unidentified Gram-negative, Gram-variable bacillus,
of bioterrorism or Gram-negative coccobacillus that only grows
on blood and chocolate agar only (not MacConkey)
Colonial ggrowth examined in biological
g safety
y is handled with extreme caution until rule-out
rule out
cabinet (while wearing gloves) until agents of
bioterrorism or highly-pathogenic agents ruled out
Blood cultures Gram staining and wet mount preparation takes
CSF cultures place in biological safety cabinet; wear gloves
Lymph node cultures
CLSI M35-A2; 2008 61 CLSI M35-A2; 2008 62
THIS IS SERIOUS ONE RULE-OUT ALGORITHM

Procedures known to create aerosols (catalase,
others) should be confined to biological safety
cabinet with extra care or avoided all together
until rule-out
Automated systems may pose danger with respect
to these organisms; may not generate accurate
identification or susceptibility data in the first place
Should not be performed until bioterrorism agent
possibility has been eliminated
J. Hosp. Infect. 22: 159-162; 1992
Diagn. Microbiol. Infect. Dis. 60: 241-246; 2008 63 Wisconsin State Laboratory of Hygiene Bench Guide 64
VITAL STATISTICS--2009 EXERCISE RULE-OUT EXERCISE--I

Sample 1: % Correct Sample 2: % Correct
Two samples sent to 116 Wisconsin laboratories Test Expected (Reporting Expected (Reporting
Result Labs) Result Labs)
101 (87%) reported data -hemolysis Negative 89.4% (85) Negative 96.3% (82)
15 did not report results -hemolysis Negative 98.9% (87) Positive 38.4% (86)
Catalase Positive 97.8% ((89)) Positive 91.0% ((89))
Sample BPE 09-2-1 Bacillus megaterium Indole Negative 100% (42) Negative 100% (44)
Sample BPE 09-2-2 Bacillus licheniformis Oxidase Negative 85.2% (54) Negative 64.8% (54)
Urease Positive 53.8% (13) Negative 76.9% (13)
Samples intended to simulate Bacillus anthracis Motility Negative 98.1% (54) Positive 94.3% (53)
Growth on Mac/EMB Negative 90.9% (77) Negative 98.7% (77)
in the context of rule-out testing
Wisconsin State Laboratory of Hygiene Audioconference; 120909 65 Wisconsin State Laboratory of Hygiene; Fall 2009 66
11
RULE-OUT EXERCISE--II RULE-OUT EXERCISE--III
Sample 1: % Correct Sample 2: % Correct Sample 1: % Correct Sample 2: % Correct
Test Expected (Reporting Expected (Reporting Test Expected (Reporting Expected (Reporting
Result Labs) Result Labs) Result Labs) Result Labs)
-hemolysis Negative 89.4% (85) Negative 96.3% (82) -hemolysis Negative 89.4% (85) Negative 96.3% (82)
-hemolysis Negative 98.9% (87) Positive 38.4% (86) -hemolysis Negative 98.9% (87) Positive 38.4% (86)
Catalase Positive 97.8% ((89)) Positive 91.0% ((89)) Catalase Positive 97.8% ((89)) Positive 91.0% ((89))
Indole Negative 100% (42) Negative 100% (44) Indole Negative 100% (42) Negative 100% (44)
Oxidase Negative 85.2% (54) Negative 64.8% (54) Oxidase Negative 85.2% (54) Negative 64.8% (54)
Urease Positive 53.8% (13) Negative 76.9% (13) Urease Positive 53.8% (13) Negative 76.9% (13)
Motility Negative 98.1% (54) Positive 94.3% (53) Motility Negative 98.1% (54) Positive 94.3% (53)
Growth on Mac/EMB Negative 90.9% (77) Negative 98.7% (77) Growth on Mac/EMB Negative 90.9% (77) Negative 98.7% (77)
Wisconsin State Laboratory of Hygiene; Fall 2009 67 Wisconsin State Laboratory of Hygiene; Fall 2009 68
THE END
Rapid identification algorithms
can benefit both the laboratory
and patient care
Requires sufficient knowledge base

in terms of knowing principles and limitations
Can (importantly) be applied to rule-out situations
Use an algorithm-based approach for bioterrorism

rule-out exercises
69
12

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OUTLINE

Overview of CLSI Document M35-A2

INTRODUCTION TO CLSI M35-A2

Utilization of these methods has resulted in

In some cases, may have resulted in cost and

INTRODUCTION TO CLSI M35-A2 BENEFITS OF RAPID RESULTS

reactions (spot testing)

MORE WARNINGS EXCLUSIONS????

P. aeruginosa, H. influenzae, Eikenella, However, possibility of serious pathogen or ability

CLSI M35-A2 PROTOCOLS

Major Players Thirteen reagents/tests

SPOT OXIDASE UREASE TUBE

Cardiobacterium hominis Cardiobacterium hominis

Eikenella corrodens Escherichia coli

Francisella tularensis Haemophilus influenzae

Haemophilus influenzae Kingella kingae

Notes LIMITATIONS/additional factors Notes

Neisseria meningitidis Neisseria lactamica

Neisseria gonorrhoeae Proteus mirabilis

Notes (if resistant to ampicillin) factors

Pseudomonas aeruginosa Pseudomonas aeruginosa

Notes Notes LIMITATIONS/additional factors

Staphylococcus aureus Staphylococcus aureus

Notes Notes LIMITATIONS/additional factors

Enterococcus spp. Aerococcus viridans

Listeria monocytogenes Listeria monocytogenes

Notes Notes LIMITATIONS/additional factors

Streptococcus anginosus group Streptococcus pneumoniae

Streptococcus pyogenes Viridans group Streptococcus

Cryptococcus neoformans ANAEROBIC GRAM-NEGATIVES

Additional tests for definitive identification

57 CLSI M35-A2; 2008 58

CLSI M35-A2; 2008 59 60

CLSI M35-A2; 2008 61 CLSI M35-A2; 2008 62

THIS IS SERIOUS ONE RULE-OUT ALGORITHM

VITAL STATISTICS--2009 EXERCISE RULE-OUT EXERCISE--I

Requires sufficient knowledge base

Can (importantly) be applied to rule-out situations

Use an algorithm-based approach for bioterrorism

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