Work-up of Respiratory Specimens Now you can breathe easier

34 th Annual Meeting Southwestern Association of Clinical Microbiology 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

Disclosures No financial disclosures No discussion of off label uses Cat and parrot mommy 2

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

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) 4

Pathogenesis of Pneumonia Aspiration of colonizing flora Inhalation of aerosols Hematologic seeding 5

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 10-10 12 CFU/mL 6

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 7

Utility of the Gram Stain 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 8

Utility of the Gram Stain Sensitivity Specificity Population Comments 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 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) Adult (CAP) Adult (HAP) Presumptive Dx in 80% Specific criteria for Gram stain

Utility of the Gram Stain Gram stain DOES NOT diagnose the presence of pneumonia Once pneumonia diagnosed Gram stain is useful in determining probable etiologic agent 10

Utility of the Gram Stain 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 11

Gram Stain Screening Criteria It all starts with a good smear Preparation Staining Standardization Consistent smear interpretation Establish quality of specimen Use interpretive comments 12

Gram Stain Screening Criteria 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 Morris et al. (1993) Zaidi and Reller (1996) Ratio of PMN to SEC Enumerate SEC/LPF and presence/absence of organisms/oif Presence/absence of organisms/oif >10 PMN/SEC >5 PMN/SEC <10 SEC/LPF and organisms present Organisms present

Gram Stain Screening Criteria 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). 14

Poor Quality 14

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% 17

Enteric-like Gram negative bacilli Report only if 10 seen per oil immersion field 18

Gram negative coccobacilli suggestive of Haemophilus Report only if 10 seen per oil immersion field YM 19

Non-enteric Gram negative bacilli Report only if 10 seen per oil immersion field YM 20

Gram negative diplococci suggestive of Moraxella Report only if 25 seen per oil immersion field 22

Gram positive cocci suggestive of Pneumococcus Report only if 25 pairs seen per oil immersion field YM 23

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

Not Routinely Reported in Respiratory Gram Stains Gram positive bacilli suggestive of Bacillus/Clostridium 26 Gram positive bacilli suggestive of Diphtheroids

NEVER Reported in Respiratory Gram Stains Yeast 27

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 Moraxella Staph 10 organisms/oif 25 organisms/oif 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 28

Mixed Flora DIRECT SMEAR SUGGESTS: 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 DIRECT SMEAR SUGGESTS: Cells: Moderate neutrophils No squamous cells Bacteria: Gram positive diplococci suggestive of Pneumococcus Mixed flora 29

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 Gram positive bacilli Yeast 30

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 31

Work up of Respiratory Cultures 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 32

Work up of Respiratory Cultures Q-Score System Q234 System Sharp, SE, et al. 2004. Cumitech 7B, Lower Respiratory Tract Infections. ASM Press, Washington, DC 33

Work up of Respiratory Cultures Q-Score System (RC Bartlett, 1974) Q-SCORE = # of potential pathogens (PP) to work up Squamous cells (-) Report value 0-1 -2-3 Key: 0 = no cells 1 = 1-9/lpf 2 = 10-24/lpf 3 = >25/lpf Neutrophils (+) 0 3 0 0 0 +1 3 0 0 0 +2 3 1 0 0 Q0 = no cult Q1 = 1PP Q2 = 2PP Q3 = 3PP +3 3 2 1 0 34

Work up of Respiratory Cultures 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) 35

Work up of Respiratory Cultures 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) 36

Work up of Respiratory Cultures 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 Work up to 2 PP if they are seen in the GS If all 3 PP are seen in the GS, MID all 3 NOTE: If mixed flora > PPs = MID PP 37

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): Work up E. coli and S. aureus MID P. aeruginosa; Report Mixed flora Q234 (3PP): 39

Example 2: Sputum 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, few Neisseria WORK UP: Q Score (Q1=1PP): Work up P. aeruginosa MID S. aureus; Report Mixed flora Q234 (2PP): 42

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): Work up E. coli and S. aureus Report Mixed flora Q234 (2PP): 45

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): Work up E. coli and S. aureus Report Mixed flora Q234 (2PP): Report Mixed flora MID E. coli and S. aureus ** ** If mixed flora > PPs = MID PP 46

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 47

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 48

Q Systems Advantages 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. 49

Q Systems Advantages 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 50

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? 51

Bronchoalveolar Lavage 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 *Fujitani and Yu, Clin Infect Dis 43 Suppl 2:S106, 2006 **Mayhall, Emerg Infect Dis 7:200, 2001 52

Bronchoalveolar Lavage Quantitative BAL Cultures Higher likelihood of pneumonia if there is at least one microorganism obtained at a concentration of 10 4 CFU/mL of lung fluid. * Commensal flora There should be no commensal flora at this site at concentrations 10 4 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) *Mayhall, Emerg Infect Dis 7:200, 2001; Baselski & Wunderink, Clin Microbiol Rev 7:533, 1994) 53

Bronchoalveolar Lavage 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

Bronchoalveolar Lavage 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 55

Bronchoalveolar Lavage Options for ORAL FLORA #1 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. #2 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

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 57

Bronchoalveolar Lavage Option #2 Example (No SEC and MF 10,000) 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 15,000 colonies/ml Coag. neg staphylococci 15,000 colonies/ml a-streptococci (not Pneumo) Add message to contact Microbiology if work up is needed 58

Bronchoalveolar Lavage Option #2 Example (No SEC and MF <10,000) Direct GS: Few PMN, No SEC, No organisms seen Culture grows 50,000 colonies/ml Enterobacter spp. 25,000 colonies/ml Klebsiella spp. 8,000 colonies/ml Coag. neg staphylococci 7,000 colonies/ml a-streptococci (not Pneumo) REPORT: 50,000 colonies/ml Enterobacter (species) with AST 25,000 colonies/ml Klebsiella (species) with AST 15,000 colonies/ml Mixed flora 59

Bronchoalveolar Lavage Option #2 Example (SEC present) Direct GS: Moderate PMN, Few SEC, Many GPC/clusters 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 60

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 61

Questions yvette.mccarter@jax.ufl.edu 62