Best Practices Workup of Wound Cultures by McCarter

34th Annual Meeting
Southwestern Association of Clinical Microbiology
Yvette McCarter, PhD, D(ABMM)
Director, Clinical Microbiology Laboratory
UF Health Jacksonville
Professor of Pathology
University of Florida College of Medicine-Jacksonville
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DISCLOSURES
 No financial disclosures
 No discussion of off-label uses
 Cat and parrot mommy
Jimi
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Logan
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OBJECTIVES
 Discuss the use of the direct Gram stain to
assess specimen quality and the advantages
of reporting morphological identifications of
organisms on direct specimen Gram stains.
 Describe the use of direct Gram stain results
to aid in the work-up of wound cultures.
 Demonstrate how to design and implement
clinically relevant, timely approaches for
cost-effective wound culture work-up.
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UF HEALTH JACKSONVILLE
620 beds
Level 1 Trauma Center
Proton Therapy Institute
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Service the underserved
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Wound Culture Work Up – It starts before the specimen gets
to the lab…
Specimen selection/
relevance
R.C. Bartlett. 1974.
Medical Microbiology:
Quality Control and
Clinical Relevance
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Use of the Gram stained smear
Timely identification
Wound culture work up
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OUT WITH THE OLD AND IN WITH THE NEW
Old
 Culture every
specimen
 Identify every
organism
 Let someone else
(the physician)
decide what to do
with the results
New
 Evaluate
appropriateness/
relevance of each
specimen
 Evaluate quality of
each specimen
 Provide clinically
relevant results that
can be interpreted
by Health Care
Provider
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SPECIMEN APPROPRIATENESS AND RELEVANCE
 Appropriate specimen sources
 Appropriate specimen volumes
 Appropriate transport
 OK, it’s a bad specimen, should I culture it
anyway?
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APPROPRIATE SPECIMEN SOURCES… NOT!
 Skin
 Superficial wounds
 Mouth
 Nose/nares
 Decubitus swabs
 Perirectal abscess
 Burden the laboratory with unnecessary effort
 Produce reports that imply infection where there is none
 Promote antimicrobial therapy of noninvasive/clinically
irrelevant bacteria
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SPECIMENS PRODUCING INFORMATION OF
DOUBTFUL VALUE
COLLECT DATE/TIME 4/11/154 0958
WOUND CULTURE SPECIMEN: Decubitus
RECEIVE DATE/TIME 4/11/15 1011
REPORT STATUS: FINAL 4/11/15
CULTURE:
Consultation requested for performance of test. Additional clinical
information is required to assure proper processing and production of
useful information from this specimen. Please consult Microbiology if
clinical considerations warrant complete processing of this specimen.
(Specimen will be held 5 days).
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APPROPRIATE SPECIMEN VOLUMES
 Swabs ---- JUST SAY
 Encourage collection of fluid/aspirate or
tissue – OPTIMAL specimen
 Education
 Limiting availability of swabs in certain
locations
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DO THE MATH…
Aerobic
Anaerobic
Fungus
AFB
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What are the chances of “cultural” success?
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EDIBLE EDUCATION
This is the specimen
you collect
This is the specimen
you send
Send us the donuts!
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APPROPRIATE SPECIMEN VOLUMES
 Why are swabs used to collect specimens?
 They are convenient
 If we’re going to get swabs…We need to
optimize the swabs we get
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THE FLOCKED SWAB
• Instant release of specimen into liquid media
• Efficiently dislodges cells to obtain cellular
material
• Improved organism recovery
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SPECIMEN TRANSPORT
 A good specimen collected appropriately is
jeopardized by inappropriate transport
 Appropriate transport device/preservation
 Optimum transport time for unpreserved
specimen = 2 hours*
 Delays
 Decreased recovery of causative organism
 Overgrowth of culture by contaminants or normal flora
Misleading results
15Miller M. 1999. A Guide to Specimen Management in Clinical Microbiology
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OK, IT’S A BAD SPECIMEN, SHOULD I
CULTURE IT ANYWAY?
Liability and the Lab
 Our job – produce accurate results in a timely
fashion for appropriate patient management
 Inappropriate specimens – producing a
result we know is inaccurate
 Admit negligence by performing test
 Disclaimer – does not insulate you from liability
 Clear, detailed rejection policy
 Review with Medical Staff
 Stick to it!
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MLO: Sept 2004, 43.
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Utility of the Gram stained smear
Abbreviated schemes for organism identification
Clinically relevant approaches for culture work-up
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UTILITY OF THE GRAM STAIN
 A well-performed direct Gram stain is rapid,
inexpensive, informational
 Allows for evaluation of specimen quality
 Identification of superficially contaminated
specimens
 Enhances the discrimination between samples
with potential pathogens vs. colonizing flora
 Provides presumptive organism identification
 Guides rational selection of preliminary antibiotic
therapy
 Guides interpretation of culture results
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UTILITY OF THE GRAM STAIN
 It all starts with a good smear…
 Preparation
 Staining
 Standardized screening criteria
 Consistent smear interpretation
 Establish quality of specimen
 Use interpretive comments
 Assist clinicians in interpreting results
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SQUAMOUS EPITHELIAL CELLS
SUPERFICIAL CONTAMINATION
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NEUTROPHILS
INFLAMMATION
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REJECTION OF POOR QUALITY WOUND
SPECIMENS???
Options…..
Do not process
Process and append
note to report
Call physician
Append message to
report
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Work up certain
organisms or
morphologically ID
all organisms
isolated
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USE OF INTERPRETIVE COMMENTS
WOUND SPECIMENS CONTAINING MORE SQUAMOUS
EPITHELIAL CELLS THAN NEUTROPHILS
X6976
Collect D/T: 1/31/2015 1215
Receive D/T: 1/31/2015 1345
Wound Culture and Gram Stain
Specimen Description Foot
Direct Smear Suggests
No neutrophils
Many squamous epithelial cells
No organisms seen
Squamous cells in this specimen indicate the presence of superficial
material that may contain contaminating or colonizing bacteria
unrelated to infection. Collection of another specimen is suggested
avoiding superficial sources of contamination.
Culture Pending
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Report Status
Preliminary
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INFLUENCE OF SPECIMEN QUALITY ON
ANAEROBIC CULTURE PROCESSING
If direct anaerobic culture requested on
appropriate specimen:
 Process specimen unless direct smear
demonstrates squamous cells
If direct anaerobic culture is not requested,
but direct smear suggests anaerobes:
Physician notified and an anaerobic culture is
requested
 Specimen processed for anaerobic culture

Laboratory determines which specimens should be
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cultured anaerobically
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USE OF INTERPRETIVE COMMENTS
SQUAMOUS EPITHELIAL CELLS ARE SEEN IN A
SPECIMEN WITH AN ANAEROBIC CULTURE REQUEST
H73026
Collect D/T: 05/01/2015 0900
1100
Receive D/T: 05/01/2015
Anaerobe Culture
Specimen Description
Leg
Evidence of superficial material. Specimen unsuitable for anaerobic
culture. Please consult Microbiology if clinical considerations
warrant complete processing of this specimen. (Specimen will be
held 5 days)
Report Status
Final 05/01/2015
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Request credited
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UTILITY OF THE GRAM STAIN
Interpretation and Reporting of Organisms in Direct Smears
Bartlett. 1982. JAMA 247:857-59
 Designation of organism genera more useful than
description of organism morphology
Bartlett et al. 1991. Diagn Microbiol Infect Dis 14:
195-201
 Reliable differentiation of Gram negative bacilli
 Bacteroides or Haemophilus – 95%
 Enteric Gram negative bacilli – 82%
 Pseudomonas – 56%
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Enteric-like Gram negative bacilli
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Plump Gram-negative rods, can have non-uniform sizes and can
sometimes
show bipolar staining
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Gram negative coccobacilli suggestive of
Haemophilus or Bacteroides
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Gram-negative coccobacilli/pleomorphic rods that may be faint
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YM
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staining
Non-enteric Gram negative bacilli
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Thin, somewhat faint staining, uniform in shape, elongated
rods,
sometimes
in pairs end to end (“hot dogs”)
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Gram positive cocci suggestive of Staphylococcus
Gram-positive spherical Gram-positive cocci in clusters or
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tetrads
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Gram positive cocci suggestive of Streptococcus
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Gram
positive cocci in pairs or chains
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Gram positive bacilli suggestive of
Gram positive bacilli suggestive of
Diphtheroids
Bacillus/Clostridium
Large box-car shaped Gram
positive
bacilli
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Small Gram positive bacilli,
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club-shaped, Chinese letter
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Yeast
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Gram positive/variable yeast with/without buds, or with/without
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pseudohyphae
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DON’T JUST REPORT WHAT YOU SEE…
INTERPRET WHAT YOU SEE AND REPORT IT
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DON’T JUST REPORT WHAT YOU SEE…
INTERPRET WHAT YOU SEE AND REPORT IT
DIRECT SMEAR SUGGESTS: DIRECT SMEAR SUGGESTS:
Cells:
Moderate neutrophils
No squamous cells
Bacteria:
Gram positive cocci in clusters
Gram positive cocci in chains
Gram positive diplococci
Cells:
Moderate neutrophils
No squamous cells
Bacteria:
Gram positive cocci suggestive
of Staphylococcus
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USE OF INTERPRETIVE COMMENTS
SPECIMENS CONTAINING NUMEROUS MORPHOTYPES OF
BACTERIA IN THE DIRECT GRAM STAIN
DIRECT SMEAR SUGGESTS:
Moderate neutrophils
No squamous cells
Gram negative rods suggestive of Enteric-like Gram negative bacilli
Gram positive cocci in clusters suggestive of Staphylococcus
Gram positive rods suggestive of Bacillus/Clostridium
Gram negative coccobacilli suggestive of Bacteroides/Haemophilus
Gram stained direct smear suggests the presence of a mixture of organisms.
Culture has not been performed because a mixed culture can be
anticipated. This information is of doubtful value for direction of therapy
against mixed infections containing this many potential pathogens. Please
consult Microbiology if clinical considerations warrant complete processing
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of this specimen. (Specimen will be held 5 days).
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ABBREVIATED SCHEMES FOR ORGANISM
IDENTIFICATION
Why do we need identify organisms rapidly?
 Value of culture results is inversely proportional
to the time it takes to report them
 Reduces delay in reporting clinically significant
isolates
 Basis for guidance of treatment with
antimicrobials
 Reduction in cost of reagents and technologist
time
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CLSI M35-A2
GRAM NEGATIVE BACTERIA
 Brucella
 Haemophilus influenzae
 Campylobacter jejuni
 Kingella kingae
 Cardiobacterium
 Moraxella catarrhalis
hominis
 Eikenella corrodens
 Escherichia coli
 Francisella tularensis
 Neisseria spp.
 Proteus spp.
 Pseudomonas
aeruginosa
CLSI M35-A2 Abbreviated Identification of Bacteria and Yeast;
Approved Guideline Second Edition, 2009
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CLSI M35-A2
GRAM POSITIVE BACTERIA AND YEAST
 Staphylococcus aureus
 Candida albicans
 Enterococcus spp.
 Candida glabrata
 Listeria monocytogenes
 Cryptococcus neoformans
 Streptococcus agalactiae
 Streptococcus anginosus gp.
 Streptococcus pneumoniae
 Streptococcus pyogenes
 Viridans group streptococci
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CLSI M35-A2
ANAEROBES
 Bacteroides fragilis
group
 Bacteroides ureolyticus
 Prevotella
intermedia/spp.
 Porphyromonas spp.
 Bilophila wadsworthia
 Fusobacterium
nucleatum
 Veillonella spp.
 Peptostreptococcus
 Clostridium difficile
 Clostridium perfringens
 Clostridium septicum
 Clostridium sordellii
 Clostridium tetani
 Propionibacterium
acnes
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CLINICALLY RELEVANT APPROACHES FOR
WOUND CULTURE WORK-UP
Raymond Bartlett 1974
“The most laboratory work and hence the
greatest cost, is associated with specimens of
the least clinical value.”
“Good bacteriology is clinically relevant
bacteriology; and clinically relevant
bacteriology cannot be performed without
making clinically relevant decisions.”
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CLINICALLY RELEVANT APPROACHES FOR
WOUND CULTURE WORK-UP
 Wound cultures are frequently contaminated
with resident flora -- difficult to determine
which organisms are potential pathogens.
 Work up can be problematic
time and
resources spent to work up cultures of little
clinical value.
 Working up mixed cultures may generate
clinically misleading results
inappropriate and unnecessary antimicrobial
therapy.
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CLINICALLY RELEVANT APPROACHES FOR
WOUND CULTURE WORK-UP
 There are no clear guidelines for working up
bacterial cultures
 Rely on information that is available in the
literature or colleagues
 Query colleagues from same or different
laboratories –often get numerous differing
opinions
 There seems to be a need for some consistency
when performing culture work up
 Uniformity in work up and reporting of bacterial
isolates
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CLINICALLY RELEVANT APPROACHES FOR
WOUND CULTURE WORK-UP
SPECIMEN QUALITY
 When working up wound cultures we are going to
assume the following:
1. Neutrophils – infection or inflammation
2. Squamous epithelial cells – superficial
contamination
 If a specimen contains a large amount of SEC,
superficial contamination is likely.
 Bacteria isolated from such specimens should
be minimally worked up
3. Extensive testing on heavily mixed cultures
should not routinely be performed
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CLINICALLY RELEVANT APPROACHES FOR
WOUND CULTURE WORK-UP
SPECIMEN QUALITY
These systems provide the means to
consistently work up organisms from wound
cultures
 Quality (Q) Score System
 Q/234 System
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WORK UP OF WOUND CULTURES
Q SCORE SYSTEM (RC BARTLETT, 1974)
 Determine average number or PMN and SEC/LPF on
direct gram stain and assign value (see Key)
 Add values together using table below to obtain Q score
 Q score = # of potential pathogens (PP) worked up
(-)
Key:
0 = no cells
1 = 1-9/lpf
2 = 1024/lpf
3 = ≥25/lpf
Q0 = 0PP
Q1 = 1PP
Q2 = 2PP
Q3 = 3PP
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WORK UP OF WOUND CULTURES
Q SCORE SYSTEM
 Specimens without SEC are considered good quality
specimens regardless of the number of PMN
 Up to 3 organisms can be considered potential
pathogens (PP) and be worked up (ID/AST) if from a
good quality specimen (Q3)
 The lower quality of the specimen (e.g., the more
SEC present) the fewer the organisms worked up
(Q2, Q1)
 Q0 – provide morphologic ID of organisms present but
no work up
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WORK UP OF WOUND CULTURES
Q SCORE SYSTEM
 # PP in culture ≤ Q-score: work up PP with ID/AST
(2PP)
(Q3)
 # PP in culture > Q-score: Look to direct Gram stain
(3PP)
(Q2)
Work up only PP that were
seen in direct Gram stain
with ID/AST
If all PP in the culture are
seen in direct Gram stain –
Do not work up; perform
morphological identification
(MID) on all isolates
MID = any rapid biochemical testing that can be performed (catalase, indole,
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oxidase, strep typing, etc.)
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WORK UP OF WOUND CULTURES
Q234 SYSTEM
 Evaluate specimen quality (PMN and SEC) using
whichever system you choose
 Culture work up is based on number of PP present:
≤ 2PP = Work up with ID/AST, as appropriate
3PP = Look to direct Gram stain
Work up to 2 PP if they are
seen in the direct
Gram stain
If all 3 PP are seen in the
direct Gram stain,
perform MID on all 3
4PP = Perform morphological identification (MID) on all isolates 49
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EXAMPLE 1: LEG WOUND
GS: moderate PMN (+2), few SEC (-1), many gram
positive cocci suggestive of Staph, many gram
positive cocci suggestive of Strep
CULT: many Staph aureus, many β hemolytic strep,
moderate coagulase neg staph, few diphtheroids
WORK UP:
Q Score (Q1=1PP):
Q234 (2PP):
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EXAMPLE 1: LEG WOUND
GS: moderate PMN (+2), few SEC (-1), many gram
positive cocci suggestive of Staph, many gram
positive cocci suggestive of Strep
CULT: many Staph aureus, many β hemolytic strep,
moderate coagulase neg staph, few diphtheroids
WORK UP:
Q Score (Q1=1PP): MID S. aureus and β Strep; Report
mixed flora
Q234 (2PP):
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EXAMPLE 1: LEG WOUND
GS: moderate PMN (+2), few SEC (-1), many gram
positive cocci suggestive of Staph, many gram
positive cocci suggestive of Strep
CULT: many Staph aureus, many β hemolytic strep,
moderate coagulase neg staph, few diphtheroids
WORK UP:
Q Score (Q1=1PP): MID S. aureus and β Strep; Report
mixed flora
Q234 (2PP): Work up S. aureus and β Strep; Report
mixed flora
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EXAMPLE 2: ABDOMINAL WOUND
GS: many PMN (+3), no SEC (0), many enteric-like
gram negative bacilli, many gram negative
coccobacilli
CULT: moderate E. coli, moderate Bacteroides spp.,
few Enterococcus spp.
WORK UP:
Q Score (Q3=3PP):
Q234 (3PP):
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EXAMPLE 2: ABDOMINAL WOUND
GS: many PMN (+3), no SEC (0), many enteric-like
gram negative bacilli, many gram negative
coccobacilli
CULT: moderate E. coli, moderate Bacteroides spp.,
few Enterococcus spp.
WORK UP:
Q Score (Q3=3PP): Work up E. coli, Bacteroides spp.
and Enterococcus spp.
Q234 (3PP):
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EXAMPLE 2: ABDOMINAL WOUND
GS: many PMN (+3), no SEC (0), many enteric-like
gram negative bacilli, many gram negative
coccobacilli
CULT: moderate E. coli, moderate Bacteroides spp.,
few Enterococcus spp.
WORK UP:
Q Score (Q3=3PP): Work up E. coli, Bacteroides spp.
and Enterococcus spp.
Q234 (3PP): Work up E. coli and Bacteroides spp.; MID
Enterococcus spp.
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PREMISE FOR Q SYSTEMS
 The more superficially contaminated the
specimen, the higher the number of colonizing
organisms present
 The quality of the specimen is important in
determining acceptability of specimen and
extent of culture work up
 If organisms are seen in the direct Gram stain,
there is a greater chance they are associated
with an infective process
 At least 105 organisms must be present to visualize
them in direct Gram stain
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ADVANTAGES OF Q SYSTEMS
 Offers a consistent approach for interpreting
cultures
o The systems are based on the quality of the
specimen (primarily SEC)
o Work up is based on organisms seen in the
direct Gram stain
o Limits the number of organisms worked up
from mixed cultures
reporting of
misleading information can be minimized
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ADVANTAGES OF Q SYSTEMS
 No potential pathogen is ever ignored
o All potential pathogens are reported – may
not be fully identified or have full AST
performed
o The pathogens that some believe should
“ALWAYS BE WORKED UP” (S. aureus, β
Strep, and P. aeruginosa) always indicated on
the report
o Either system can be modified to include
screening for MRSA, VRE, ESBLs, etc
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ADVANTAGES OF Q SYSTEMS
 They provide guidelines
o Both Q Systems offer guidelines for a
systematic approach to culture interpretation
and work up
o These guidelines are just that = Guidelines!
Exceptions can be made if necessary
o Any concerned physician can consult with
microbiology to have further work performed
on any culture if clinically indicated
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THE Q SYSTEMS IN PRACTICE…
C Matkoski, SE Sharp, and DL Kiska. 2006.
Evaluation of the Q Score and Q234 Systems for CostEffective and Clinically Relevant Interpretation of
Wound Cultures.
J Clin Microbiol 44:1869-1872
Reagent cost savings
Reduced unnecessary culture work-up
Allowed technologists to make more independent
and consistent decisions about the significance of
organisms in a wound culture
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“Your scientists were so preoccupied
with whether or not they could, they
didn't stop to think if they should.”
-Dr. Ian Malcolm
Jurassic Park
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BEST PRACTICES IN THE WORK UP OF
WOUND CULTURES
 What happens to the specimen before you get
it is important
 Avoid culturing inappropriate specimens
 If you’ve got to get a swab… Get a flocked swab
 Master the Gram stain and make the most of it
 The value of culture results is inversely
proportional to the time it takes to report it
 Employ methods for rapid organism identification
 Watch your P’s and Q’s
 Determine your “P’s” (potential pathogens)
 Pick one of the “Q’s” (Q systems)
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January 29-30, 2016
Renaissance Resort at World
Golf Village
St. Augustine, Florida
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