Work-up of Respiratory Specimens… - SWACM - …€¦ · CULT: many P. aeruginosa, moderate Staph...

Work-up of Respiratory Specimens… Now you can breathe easier

Yvette S. 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

Disclosures

No financial disclosures

No discussion of off label uses

Cat and parrot mommy

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Objectives

Discuss the value of the gram stained smear as a reliable rapid diagnostic tool and criteria for assessing specimen quality by microscopic screening.

Describe recognition and reporting of organisms by genera rather than organism morphology.

Discuss the criteria for and use of “mixed flora” in respiratory gram stains.

Describe the Q score and Q234 systems and how they can be used to guide work up of lower respiratory tract cultures.

Discuss the appropriate work up of bronchoalveolar lavage specimens. 3

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The major goal of the clinical microbiology laboratory is to provide information of maximal clinical or epidemiological usefulness as rapidly as is consistent with acceptable accuracy and minimal cost.

Jay P. Sanford, MD (1974)

The culture of lower respiratory specimens may result in more unnecessary microbiologic effort than any other type of specimen.

Raymond C. Bartlett, MD (1974)

Pathogenesis of Pneumonia

Aspiration of colonizing flora

Inhalation of aerosols

Hematologic seeding

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http://en.wikipedia.org/wiki/File:PneumonisWedge09.JPG

Oral Flora or Potential Pathogen?

Oral Flora

Corynebacterium spp.

Coagulase negative staphylococci

Staphylococcus aureus

Neisseria spp.

Haemophilus influenzae

Streptococcus pneumoniae

Moraxella catarrhalis

Gram negative bacilli

Potential Pathogens

Staphylococcus aureus

Haemophilus influenzae

Streptococcus pneumoniae

Moraxella catarrhalis

Gram negative bacilli

6 Oral flora – 1010-1012 CFU/mL

Utility of the Gram Stain

Rapid, inexpensive, informational

Evaluation of specimen quality

Identify superficially contaminated specimens

Enhance discrimination between samples with potential pathogens vs. colonizing flora

Presumptive organism ID

Guide rational selection of preliminary antibiotic therapy

Guides interpretation of culture results

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Majority of the literature supports the clinical usefulness of gram stained sputum smears

Wide range in reported sensitivity (35-96% and specificity (12-85%)

Reference standard – sputum culture

Variable care in specimen collection – “Good quality” specimen

Multiple criteria for assessing Gram stain smears

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Sensitivity Specificity Population Comments

Cao et al. J Infect Chemother 2004

Spn: 81% Hflu: 86% Mcat: 91%

Spn: 98% Hflu: 95% Mcat: 98%

Pediatric

Musher et al. CID 2004

Spn: 57% NA Adult Included pts on antibiotics

Rosón et al.

CID 2000 Spn: 57% Hflu: 82%

Spn: 97% Hflu: 99%

Adult (CAP) Presumptive Dx in 80%

Anelavis et al. J Infect 2009

Spn: 82% Hflu: 79% Saur: 76% GNR: 78%

Spn: 93% Hflu: 96% Saur: 96% GNR: 95%

Adult (CAP) Specific criteria for Gram stain

Blot et al. Am J Respir Crit Care Med 2000

EA: 91% PTC: 70%

EA: 64% PTC: 96%

Adult (HAP)


Gram stain DOES NOT diagnose the presence of pneumonia

Once pneumonia diagnosed Gram stain is useful in determining probable etiologic agent

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Heineman et al. 1977. J Clin Microbiol 6:518-27

50% of the information gleaned from sputum cultures is clinically misleading in the absence of correlation with direct gram stain results

Gleckman et al. 1988. J Clin Microbiol 26:846-49

Selection of appropriate monotherapy 94% of the time when guided by bacterial morphotypes from the gram stain

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Gram Stain Screening Criteria

It all starts with a good smear…

Preparation

Staining

Standardization

Consistent smear interpretation

Establish quality of specimen

Use interpretive comments

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Author (year) Method Minimum criteria

Bartlett (1974) Sum of PMN/LPF (10-25, 1+; >25, 2+), mucus (1+); SEC (10-25, -1; >25, -2)

Score of >0

Murray and Washington (1975) Geckler et al. (1977)

Enumerate SEC/LPF <10 SEC/LPF <25 SEC/LPF

Van Scoy (1977) Enumerate PMN/LPF

>25 PMN/LPF

Heineman and Radano (1979) Kalin et al. 1983

Ratio of PMN to SEC >10 PMN/SEC >5 PMN/SEC

Morris et al. (1993) Enumerate SEC/LPF and presence/absence of organisms/OIF

<10 SEC/LPF and organisms present

Zaidi and Reller (1996) Presence/absence of organisms/OIF

Organisms present


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COLLECT DATE/TIME 2/10/15 1655 RESP CULTURE SPECIMEN: Sputum

RECEIVE DATE/TIME 2/10/15 1800

REPORT STATUS: FINAL 2/10/15

DIRECT SMEAR SUGGESTS:

No neutrophils

Many squamous cells

Not representative of lower respiratory tract secretions. Culture not

performed. Please consult Microbiology if clinical considerations

warrant complete processing of this specimen. (Specimen will be

held 5 days).

Poor Quality

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Good Quality

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

Report only if ≥ 10 seen per oil immersion field

19 YM

Gram negative coccobacilli suggestive of Haemophilus


20 YM

Non-enteric Gram negative bacilli


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Gram negative diplococci suggestive of Moraxella


23 YM

Gram positive cocci suggestive of Pneumococcus

Report only if ≥ 25 pairs seen per oil immersion field

Gram positive cocci suggestive of Staphylococcus


Not Routinely Reported in Respiratory Gram Stains

Gram positive cocci suggestive of Streptococcus

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Not Routinely Reported in Respiratory Gram Stains

Gram positive bacilli suggestive of

Bacillus/Clostridium

Gram positive bacilli suggestive of Diphtheroids

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NEVER Reported in Respiratory Gram Stains

Yeast

“Mixed Flora”

Used only with respiratory specimens

Use of objective criteria (# of organisms present per OIF) to distinguish resident flora or colonizers from potential pathogens:

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Morphology Call if:

Gram negative bacilli ≥ 10 organisms/OIF

Moraxella ≥ 25 organisms/OIF

Staph ≥ 50 organisms/OIF

S. pneumoniae ≥25 pairs/OIF

Bartlett 1982 JAMA Wright et al. 1990 Am J Med Normandin et al. 1997 ASM C-91

“Mixed Flora”


Cells:

Moderate neutrophils

No squamous cells

Bacteria:

Few Gram negative rods

Many Gram positive diplococci

Moderate Gram negative diplococci

Few Gram positive rods

Few Gram negative coccobacilli

Rare Gram positive cocci in clusters

Few yeast


Cells:

Moderate neutrophils

No squamous cells

Bacteria:

Gram positive diplococci suggestive of Pneumococcus

Mixed flora

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Mixed Flora Criteria

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Streptococcus

Gram positive bacilli

Yeast

Morphology Call if:

Gram negative bacilli ≥ 10 organisms/OIF

Moraxella ≥ 25 organisms/OIF

Staph ≥ 50 organisms/OIF

S. pneumoniae ≥25 pairs/OIF

Work up of Respiratory Cultures

There are no clear guidelines for working up bacterial cultures

Literature

Colleagues

There seems to be a need for some consistency when performing culture work up

Uniformity in work up and reporting of bacterial isolates

When to perform AST

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Specimen Quality

Premise:

PMN are an indication of infection or inflammation

SEC indicate superficial contamination

If a specimen contains a large amount of SEC, superficial contamination is likely

The specimen should be recollected

Extensive testing on heavily mixed cultures should not routinely be performed

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Q-Score System

Q234 System

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Sharp, SE, et al. 2004. Cumitech 7B, Lower Respiratory Tract Infections. ASM Press, Washington, DC


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Key:

0 = no cells

1 = 1-9/lpf

2 = 10-24/lpf

3 = >25/lpf

Q-SCORE = # of potential pathogens (PP) to work up

Q0 = no cult

Q1 = 1PP

Q2 = 2PP

Q3 = 3PP

Q-Score System (RC Bartlett, 1974)

Squamous cells (-)

Report value 0 -1 -2 -3

0 3 0 0 0

+1 3 0 0 0

Neutrophils (+) +2 3 1 0 0

+3 3 2 1 0


“Q-Score” System

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)

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“Q-Score” System

# PP in culture < Q-score: work up PP with ID/AST

(2PP) (Q3)

# PP in culture > Q-score: Look to Gram stain

(3PP) (Q2)

Work up PP that were seen in Gram stain with ID/AST

If all PP in the culture are seen in Gram stain = do not work up; perform morphological identification (MID)

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Q234 System (SE Sharp, 2002)

Gram stain Quality Check: PMN & SEC

Reject any sputum for culture according to normal protocol

Culture work up is based on number of PP present: 2 PP = Work up (< 2 PP)

4 PP = MID

3 PP = Look to Gram stain

37 NOTE: If mixed flora > PPs = MID PP

Work up to 2 PP if they are seen in the GS

If all 3 PP are seen in the GS, MID all 3

Example 1: Sputum

GS: many PMN (+3), few SEC (-1), many enteric-like gram negative bacilli, many gram positive cocci suggestive of Staph, few Mixed flora (yeast)

CULT: moderate P. aeruginosa, moderate E.coli, moderate Staph aureus, few yeast

WORK UP: Q Score (Q2=2PP): Q234 (3PP):

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Example 1: Sputum



WORK UP: Q Score (Q2=2PP): Work up E. coli and S. aureus

MID P. aeruginosa; Report Mixed flora Q234 (3PP):

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Example 1: Sputum



WORK UP: Q Score (Q2=2PP): Work up E. coli and S. aureus

MID P. aeruginosa; Report Mixed flora

Q234 (3PP): Work up E. coli and S. aureus MID P. aeruginosa; Report Mixed flora

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Example 2: Sputum

GS: many PMN (+3), moderate SEC (-2), many nonenteric-like gram negative bacilli, moderate Mixed flora

CULT: many P. aeruginosa, moderate Staph aureus, few viridans Strep, few Neisseria

WORK UP: Q Score (Q1=1PP):

Q234 (2PP):

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Example 2: Sputum



WORK UP: Q Score (Q1=1PP): Work up P. aeruginosa

MID S. aureus; Report Mixed flora

Q234 (2PP):

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Example 2: Sputum



WORK UP: Q Score (Q1=1PP): Work up P. aeruginosa

MID S. aureus; Report Mixed flora

Q234 (2PP): Work up P. aeruginosa and S. aureus Report Mixed flora

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Example 3: Tracheal Aspirate

GS: many PMN (+3), few SEC (-1), many Mixed flora (few enteric-like GNB; moderate gram positive cocci suggestive of Staph)

CULT: moderate diphtheroids, moderate coag negative Staph, few E.coli, rare Staph aureus

WORK UP: Q Score (Q2=2PP):

Q234 (2PP):

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WORK UP: Q score (Q2=2PP): Work up E. coli and S. aureus

Report Mixed flora Q234 (2PP):

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WORK UP: Q-Score (Q2=2PP): Work up E. coli and S. aureus

Report Mixed flora Q234 (2PP): Report Mixed flora

MID E. coli and S. aureus **

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** If mixed flora > PPs = MID PP

Premise for “Q” Systems

Based on published prevalence of potential pathogen colonization of the oropharynx

The more superficially contaminated the specimen, the higher the # of colonizing organisms present

Quality of specimen is important in determining acceptability of specimen and extent of culture work up

If organisms seen in smear, greater chance they are associated with an infective process

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“Q” Systems Advantages

Offers a consistent approach for interpreting cultures

Based on specimen quality (primarily SEC)

Based on organisms seen in Gram stain (organism seen on smear should be in a significant number in the specimen, ≥ 105/mL)

Limits number of organisms worked up from mixed cultures reporting of misleading information minimized

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No Potential Pathogen is ever ignored

All potential pathogens reported (may not perform full ID/AST)

Pathogens that some believe should “ALWAYS BE WORKED UP” (S. aureus, b-strep, P. aeruginosa ) are identified and always indicated on the report

Can be modified to include screening for MRSA, VRE, etc.

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Guidelines

The Q Systems offer guidelines for a systematic approach to culture interpretation

These Guidelines are just that = Guidelines! Exceptions can be made

Any concerned physician can consult with microbiology to have further work performed on any culture if clinically indicated

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Bronchoalveolar Lavage

What to do with the BAL…

Quantitate or not

What quantity to work up

What to do with that ‘resident flora’

When to do susceptibility testing

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?


Quantitative cultures of BAL specimens are imperfect predictors of the presence of pneumonia in mechanically-ventilated patients.*

Quantitative cultures have a reported sensitivity and specificity of 91% and 78%, respectively.**

Despite the limitations, there are no alternatives that are clearly better

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*Fujitani and Yu, Clin Infect Dis 43 Suppl 2:S106, 2006 **Mayhall, Emerg Infect Dis 7:200, 2001


Quantitative BAL Cultures

Higher likelihood of pneumonia if there is “at least one microorganism obtained at a concentration of ≥ 104 CFU/mL of lung fluid.”*

Commensal flora

There should be no commensal flora at this site at concentrations ≥104 CFU/mL

Commensal oral flora, when aspirated, can cause pneumonia

Quantitation is used to determine whether this has occurred or not

There should also be NO squamous epithelial cells present (this represents oral contamination)

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*Mayhall, Emerg Infect Dis 7:200, 2001; Baselski & Wunderink, Clin Microbiol Rev 7:533, 1994)


IDSA/ATS guidelines for VAP "Significant growth of oropharyngeal commensals

(viridans group streptococci, coagulase-negative staphylococci, Neisseria spp., and Corynebacterium spp.) from distal bronchial specimens is difficult to interpret…,

but these organisms can produce infection in

immunocompromised hosts and some immunocompetent patients.”

Am J Respir Crit Care Med 171:388, 2005 54


Options for POTENTIAL PATHOGENS ≥ Cutoff Quantitate and perform ID/AST on potential pathogens if ≥10,000

for BAL and ≥1,000 for Brush using Q score or Q234 systems Quantitate and perform ID/AST on up to 3 potential pathogens if

≥10,000 for BAL and ≥ 1000 for Brush (if > 3 report MID) Option for POTENTIAL PATHOGENS < Cutoff Quantitate and report MID on any potential pathogens <10,000 for

BAL and < 1,000 for Brush

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Options for ORAL FLORA Quantitate total amount and report as Mixed flora If no SEC were seen on initial GS, for each OF isolate at ≥10,000

for BAL and ≥1,000 for Brush, report MID.

If no SEC were seen on initial GS, for each OF isolate at <10,000 for BAL and <1,000 for Brush, quantitate and report TOTAL AMOUNT of combined oral flora’(i.e. 7000 diphtheroids+ 8000 non hemolytic strep = 15,000 Mixed flora)

If SEC were seen on initial GS (= contamination), quantitate and

report Mixed flora regardless of amount present 56

#1

#2

Bronchoalveolar Lavage Oral Flora Option #1 Example

Direct GS: Few PMN, No SEC, No organisms seen

Culture grows

50,000 colonies/mL Staphylococcus aureus

15,000 colonies/mL Coag. neg staphylococci

15,000 colonies/mL a-streptococci (not Pneumo)

REPORT:

50,000 colonies/mL S. aureus with AST

30,000 colonies/mL Mixed flora

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Bronchoalveolar Lavage Option #2 Example (No SEC and MF ≥10,000)


Culture grows




REPORT:




58 Add message to contact Microbiology if work up is needed

Bronchoalveolar Lavage Option #2 Example (No SEC and MF <10,000)


Culture grows

50,000 colonies/mL Enterobacter spp.

25,000 colonies/mL Klebsiella spp.



REPORT:

50,000 colonies/mL Enterobacter (species) with AST

25,000 colonies/mL Klebsiella (species) with AST


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Bronchoalveolar Lavage Option #2 Example (SEC present)

Direct GS: Moderate PMN, Few SEC, Many GPC/clusters

Culture grows




REPORT:



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Conclusions

Gram stain of direct respiratory specimens is useful

DO IT WELL and USE IT

Specimen quality assessment

Rapid presumptive identification

Guide culture work up

Mind your P’s and Q’s

Determine your “P’s” (potential pathogens)

Pick one of the “Q’s” (Q systems)

Report consistent and clinically-relevant respiratory

culture results

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Questions

62 [email protected]

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