Clinically Relevant, Cost-Effective Microbiology

ASM 2011 Clinical Core Curriculum III 

Best Practices in the Work Up 

of Respiratory Cultures 

Yvette S. McCarter, PhD, D(ABMM) 

Director, Clinical Microbiology Laboratory 

Shands Jacksonville 

Professor of Pathology 

University of Florida College of Medicine-Jacksonville

Objectives 

Discuss the value of the gram stained smear as a 

reliable rapid diagnostic tool. 

Discuss 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 cost-effective, clinically-relevant strategies 

for laboratory work up of lower respiratory tract 

specimens. 

2

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) 

3

Pathogenesis of Pneumonia 

Aspiration of colonizing flora 

Inhalation of aerosols 

Hematologic seeding 

4

Oral Flora or Potential Pathogen? 

Oral Flora 

Corynebacterium spp. 

Coagulase negative 

staphylococci 

Staphylococcus aureus 

Neisseria spp. 

Haemophilus influenzae 

Streptococcus 

pneumoniae 

Moraxella catarrhalis 

Potential Pathogens 

Staphylococcus aureus 

Haemophilus influenzae 

Streptococcus 

pneumoniae 

Moraxella catarrhalis 

Gram negative bacilli 

Gram negative bacilli 

Oral flora – 10 10-1012 CFU/mL 

5

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 

6


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 

7

Cao et al. 

J Infect 

Chemother 

2004 

Musher et al. 

CID 2004 

Rosón et al. 

CID 2000 

Anelavis et al. 

J Infect 2009 

Blot et al. 

Am J Respir Crit 

Care Med 2000 


Sensitivity Specificity Population Comments 

Spn: 81% 

Hflu: 86% 

Mcat: 91% 

Spn: 98% 

Hflu: 95% 

Mcat: 98% 

Pediatric 

Spn: 57% NA Adult Included pts 

on antibiotics 

Spn: 57% 

Hflu: 82% 

Spn: 82% 

Hflu: 79% 

Saur: 76% 

GNR: 78% 

EA: 91% 

PTC: 70% 

Spn: 97% 

Hflu: 99% 

Spn: 93% 

Hflu: 96% 

Saur: 96% 

GNR: 95% 

EA: 64% 

PTC: 96% 

Adult (CAP) Presumptive 

Dx in 80% 

Adult (CAP) Specific 

criteria for 

Gram stain 

Adult (HAP)


Gram stain DOES NOT diagnose the presence 

of pneumonia 

Once pneumonia diagnosed Gram stain is 

useful in determining probable etiologic agent 

9


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 

10

IDSA/ATS 

Consensus Guidelines on the 

Management of CAP 

Sputum cultures may have a major impact on the 

care of an individual patient 

Antibiotic susceptibility patterns 

Pretreatment Gram stain and culture of expectorated 

sputum should be performed only if a good-quality 

specimen can be obtained 

The benefit of a sputum Gram stain is 2-fold: 

Broadens initial empirical coverage for less common 

etiologies (S. aureus or gram-negative bacilli) 

Validates subsequent sputum culture results 

Mandell et al. 2007 CID 44 (Suppl 2):S27-S72 

11

IDSA/ATS 

Consensus Guidelines on the 

Management of HAP, VAP and HCAP 

Reliable tracheal aspirate Gram stain 

Direct initial empiric antimicrobial therapy 

May increase the diagnostic value of the clinical 

pulmonary infection score 

Culture results are not available immediately 

Gram stain can be used to increase the likelihood of 

a subsequent positive culture and guide antibiotic 

therapy before culture results 

Less use of antibiotics with no adverse outcomes 

Improved mortality 

Neiderman, Craven et al. 2005. Am J Respir Crit Care Med 171:388-416 

12

Gram Stain Screening Criteria 

Standardization 

Consistent smear interpretation 

Establish quality of specimen 

Optimal smear preparation and staining 

When not to apply the criteria 

Legionella culture 

Use interpretive comments 

13


COLLECT DATE/TIME 2/10/11 1655 RESP CULTURE SPECIMEN: Sputum 

RECEIVE DATE/TIME 2/10/11 1800 

DIRECT SMEAR SUGGESTS: 

No neutrophils 

Many squamous epithelial cells 

REPORT STATUS: FINAL 2/10/11 

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). 

12


Author (year) Method Minimum criteria 

Bartlett (1974) Sum of PMN/LPF (10-25, 

1+; >25, 2+), mucus (1+); 

SEC (10-25, -1; >25, -2) 

Murray and Washington 

(1975) 

Geckler et al. (1977) 

Score of >0 

Enumerate SEC/LPF 10 PMN/SEC 

Morris et al. (1993) Enumerate SEC/LPF and 

presence/absence of 

organisms/OIF 

Zaidi and Reller (1996) Presence/absence of 

organisms/OIF 

>5 PMN/SEC 

Poor Quality 

14 14

Good Quality 

15

“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: 

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 

18


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 

“Mixed Flora” 


Cells: 

Moderate neutrophils 

No squamous cells 

Bacteria: 

Gram positive diplococci suggestive 

of Pneumococcus 

Mixed flora 

19

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% 

20

Enteric-like Gram negative bacilli 

Report only if > 10 seen per oil immersion field 

21

Gram negative coccobacilli suggestive of Haemophilus 


YM 

22

Non-enteric Gram negative bacilli 


YM 

23

Gram negative diplococci suggestive of Moraxella 


25

Gram positive cocci suggestive of Pneumococcus 

Report only if > 25 pairs seen per oil immersion field 

YM 

26

Gram positive cocci suggestive of Staphylococcus 

Report only if > 50 seen per oil immersion field

Not Routinely Reported in Respiratory Gram Stains 

Gram positive cocci suggestive of Streptococcus


Gram positive bacilli 

suggestive of 

Bacillus/Clostridium 

29 


suggestive of Diphtheroids


Yeast 

30

Mixed Flora Criteria 

Morphology Call if: 

Gram negative bacilli ≥ 10 organisms/OIF 

Moraxella ≥ 25 organisms/OIF 

Staph ≥ 50 organisms/OIF 

S. pneumoniae ≥25 pairs/OIF 

Streptococcus 


Yeast 

31

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 

32


Premise: 

Specimen Quality 

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 

33


Q-Score System 

Q234 System 

PMN-association System 

Sharp, SE, et al. 2004. Cumitech 7B, Lower Respiratory Tract 

Infections. ASM Press, Washington, DC 

34


Key: 

0 = no cells 

1 = 1-9/lpf 

2 = 10-24/lpf 

3 = >25/lpf 

Q-Score System (RC Bartlett, 1974) 

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

Report value 

Neutrophils (+) 

Squamous cells (-) 

0 -1 -2 -3 

0 3 0 0 0 

+1 3 0 0 0 

+2 3 1 0 0 

+3 3 2 1 0 

Q0 = no cult 

Q1 = 1PP 

Q2 = 2PP 

Q3 = 3PP 

35


“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) 

36


“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) 

37


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 

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 

38


PMN-Association System 

Success in microscopic evaluation relies on strict 

cytological criteria 

>25 PMN, < 10 SEC 

>25 PMN, 10 PMN per SEC (10:1 ratio) 

Evaluate the presence of predominant 

morphotypes associated with WBC 

1 morphotype 10 organisms/OIF 39


PMN-Association System 

Quantitation of organisms in smears 

inconsistent and inaccurate 

Technologist variability 

Do not rely on quantitation to determine 

relatedness to infection PMN more 

predictive 

Work up organisms seen in association with 

PMN 

40

Example 1: Sputum 

GS: many PMN (+3), few SEC (-1), many enteric-like gram 

negative bacilli, moderate 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): 

41







WORK UP: 

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

MID P. aeruginosa; Report Mixed flora 

Q234 (3PP): 

42







WORK UP: 

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


Q234 (3PP): Work up E. coli and S. aureus 


43


GS: many PMN (+3), moderate SEC (-2), many nonentericlike 

gram negative bacilli, moderate Mixed flora 

CULT: many P. aeruginosa, moderate Staph aureus, few 

viridans Strep 

WORK UP: 


Q234 (2PP): 

44






WORK UP: 

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

MID S. aureus; Report Mixed flora 

Q234 (2PP): 

45






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 

46

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: 


Q234 (2PP): 

47







WORK UP: 

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


Q234 (2PP): 

48







WORK UP: 

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


Q234 (2PP): Report Mixed flora 

MID E. coli and S. aureus ** 

** If mixed flora > PPs = MID PP 

49

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 

50

“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, >10 5 /mL) 

Limits number of organisms worked up from 

mixed cultures reporting of misleading 

information minimized 

51


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, -strep, P. 

aeruginosa ) are identified and always 

indicated on the report 

Can be modified to include screening for 

MRSA, VRE, etc. 

52


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 

53

References 

Sharp, SE, et al. 2004. Cumitech 7B, Lower 

Respiratory Tract Infections. ASM Press, 

Washington, DC 

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. 

54

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 

Watch 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 

55

Questions 

yvette.mccarter@jax.ufl.edu 

56

Clinically Relevant, Cost-Effective Microbiology

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