34 Annual Meeting Southwestern Association of Clinical ... · 1 Yvette McCarter, PhD, D(ABMM) Director, Clinical Microbiology Laboratory UF Health Jacksonville Professor of Pathology

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Yvette McCarter, PhD, D(ABMM)

Director, Clinical Microbiology Laboratory

UF Health Jacksonville

Professor of Pathology

University of Florida College of Medicine-Jacksonville

34th Annual Meeting Southwestern Association of Clinical Microbiology

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DISCLOSURES

No financial disclosures

No discussion of off-label uses

Cat and parrot mommy

2 Jimi 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

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620 beds

Level 1 Trauma Center

Proton Therapy Institute

Service the underserved

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Specimen selection/ relevance

R.C. Bartlett. 1974.

Medical Microbiology:

Quality Control and

Clinical Relevance

Use of the Gram stained smear Timely identification

Wound culture work up

Wound Culture Work Up – It starts before the specimen gets to the lab…

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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

Mouth

Decubitus swabs

Perirectal abscess

Superficial wounds

Nose/nares

Burden the laboratory with unnecessary effort

Produce reports that imply infection where there is none

Promote antimicrobial therapy of noninvasive/clinically irrelevant bacteria 8

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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…

11 What are the chances of “cultural” success?

Aerobic

Anaerobic

Fungus

AFB

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EDIBLE EDUCATION

This is the specimen you collect

This is the specimen you send

Send us the donuts! 12

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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

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Misleading results

Miller 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?

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!

MLO: Sept 2004, 43. 16

Liability and the Lab

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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 18

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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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NEUTROPHILSINFLAMMATION

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REJECTION OF POOR QUALITY WOUND SPECIMENS???

Do not process

Call physician

Append message to report

Process and append note to report

Work up certain organisms or

morphologically ID all organisms

isolated

Options…..

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USE OF INTERPRETIVE COMMENTS WOUND SPECIMENS CONTAINING MORE SQUAMOUS EPITHELIAL CELLS THAN NEUTROPHILS

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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

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

cultured anaerobically 24

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USE OF INTERPRETIVE COMMENTS SQUAMOUS EPITHELIAL CELLS ARE SEEN IN A SPECIMEN WITH AN ANAEROBIC CULTURE REQUEST

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H73026

Collect D/T: 05/01/2015 0900 Receive D/T: 05/01/2015

1100

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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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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Interpretation and Reporting of Organisms in Direct Smears

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Plump Gram-negative rods, can have non-uniform sizes and can

sometimes show bipolar staining

Enteric-like Gram negative bacilli

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Gram negative coccobacilli suggestive of Haemophilus or Bacteroides

Gram-negative coccobacilli/pleomorphic rods that may be faint

staining

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Thin, somewhat faint staining, uniform in shape, elongated rods,

sometimes in pairs end to end (“hot dogs”)

Non-enteric Gram negative bacilli

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Gram positive cocci suggestive of Staphylococcus

Gram-positive spherical Gram-positive cocci in clusters or

tetrads

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Gram positive cocci suggestive of Streptococcus

Gram positive cocci in pairs or chains

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Gram positive bacilli suggestive of Bacillus/Clostridium

Gram positive bacilli suggestive of Diphtheroids

Large box-car shaped Gram

positive bacilli

Small Gram positive bacilli,

club-shaped, Chinese letter

appearance

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Yeast

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Gram positive/variable yeast with/without buds, or with/without

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:

Cells:

Moderate neutrophils

No squamous cells

Bacteria:

Gram positive cocci in clusters

Gram positive cocci in chains

Gram positive diplococci


Cells:


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

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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

of this specimen. (Specimen will be held 5 days). 36

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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

Campylobacter jejuni

Cardiobacterium hominis

Eikenella corrodens

Escherichia coli

Francisella tularensis

Haemophilus influenzae

Kingella kingae

Moraxella catarrhalis

Neisseria spp.

Proteus spp.

Pseudomonas aeruginosa

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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

Enterococcus spp.

Listeria monocytogenes

Streptococcus agalactiae

Streptococcus anginosus gp.

Streptococcus pneumoniae

Streptococcus pyogenes

Viridans group streptococci

Candida albicans

Candida glabrata

Cryptococcus neoformans

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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

“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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Raymond Bartlett 1974

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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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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

Quality (Q) Score System

Q/234 System

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These systems provide the means to

consistently work up organisms from wound

cultures

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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

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Q0 = 0PP

Q1 = 1PP

Q2 = 2PP

Q3 = 3PP

(-)

Key:

0 = no cells

1 = 1-9/lpf

2 = 10-

24/lpf

3 = ≥25/lpf

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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)

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If all PP in the culture are

seen in direct Gram stain –

Do not work up; perform

morphological identification

(MID) on all isolates

Work up only PP that were

seen in direct Gram stain

with ID/AST

MID = any rapid biochemical testing that can be performed (catalase, indole,

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

4PP = Perform morphological identification (MID) on all isolates

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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

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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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WORK UP:

Q Score (Q1=1PP): MID S. aureus and β Strep; Report mixed flora

Q234 (2PP):

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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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WORK UP: Q Score (Q3=3PP): Work up E. coli, Bacteroides spp.

and Enterococcus spp. Q234 (3PP):

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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. 55

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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 56

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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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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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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 Cost-Effective 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) 62

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34 Annual Meeting Southwestern Association of Clinical ... · 1 Yvette McCarter, PhD, D(ABMM) Director, Clinical Microbiology Laboratory UF Health Jacksonville Professor of Pathology

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