References Required document for Laboratory Accreditation by the College of American Pathologists.

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1 Subject Culture: Lower Respiratory Tract Index Number Lab-3375 Section Laboratory Subsection Microbiology Category Departmental Contact Sarah Stoner Last Revised 9/11/2017 References Required document for Laboratory Accreditation by the College of American Pathologists. Applicable To Employees of Gundersen Health System laboratories. Detail Specimens from the lower respiratory tract are plated to a standard set of media for the recovery of potential pathogens. CLINICAL SIGNIFICANCE: Infections of the lower respiratory tract are a major cause of morbidity and mortality. Diagnosis of these infections frequently is complicated by the contamination of specimens with upper respiratory tract secretions during collection. Because the upper respiratory tract may be colonized with potential pathogens not involved in the infection of the lower tract, and may yield organisms capable of inhibiting the bacteria involved in the lower tract pathology, the laboratory should ensure that an appropriate specimen is processed. The specimen must be microscopically examined both to assess its quality and to look for organisms associated with an inflammatory response. SPECIMEN: 1. Sputum 2. Tracheal aspirates 3. Bronchial washings 4. Bronchial brushes 5. Bronchial biopsy 6. Bronchial alveolar lavage 7. Trans-tracheal aspirates 8. Lung aspirates 9. Lung biopsy Refer to specimen collection policy for instruction on specimen collection Lab-1215 Microbiology Specimen Collection and Transport. NOTES: 1. Sputum may not be the specimen of choice for determining the etiologic agent of bacterial pneumonia. Blood specimens, lavage specimens or trans-tracheal aspirates may be more ac curate. Page 1 of 7

2 2. Lower respiratory tract secretions from infected patients are confirmed by noting the presence of large numbers (>25 cells per low power field) of leukocytes in the absence (<25 cells per low power field) of epithelial cells. Since epithelial cells in the specimen signal gross contamination with oropharyngeal flora, the required number of WBC's must be present to culture. 3. Careful attention to the instructions given to the patient will greatly reduce the number of inappropriate specimens. 4. The first, early morning specimen is preferred. Pooled specimens are not recommended for culture. REAGENTS / MATERIALS: 1. 5% Sheep Blood Agar Plate (BAP) 2. MAC plate 3. PEA plate 4. Chocolate Agar plate 5. Thio Broth (for aspirates and biopsy specimens obtained by invasive techniques.) 6. Glass slide EQUIPMENT / INSTRUMENTATION: 5% CO2 Incubator 33 C-37 C Inoculating loop QUALITY CONTROL: Refer to Lab-3257 User Quality Assurance Procedure for Culture Media. Specimen quality is monitored by the Gram stain. Specimens of poor quality are recollected if possible. See Lab 3279 Gram stain and the following section on reporting Gram stains. Implementation Pick the most purulent part of the specimen and sample with sterile cotton-tipped applicator stick. Inoculate BAP, CHOC, MAC and PEA plates. Make a smear for Gram stain. 1. Perform Gram stain and report results. Refer to Interpretation section of this policy to evaluate sputum specimen quality and request a new specimen if necessary. 2. Streak plates for isolation and place a P disc in the first (heaviest inoculum) quadrant. 3. Incubate the plates overnight at C in the CO 2 incubator. 4. Examine the plates after 24 and 48 hours of incubation. Correlate Gram stain and culture to determine significant isolates. TABLE 1 Specimen Sputum coughed or induced Endotracheal tube aspirate NOT likely significant Likely significant Potential pathogen not seen on gram stain and only small to moderate growth on culture. Low PMN s Seen in gram stain and culture. Abundant PMNs Potential pathogen not seen on gram stain and only small to moderate growth on culture. Low PMN s Seen in gram stain and culture. BAL Potential pathogen not seen on gram stain. Potential pathogen seen in every 100x Page 2 of 7

3 Additional information suggest Significance Intracellular bacteria (potential pathogens) seen on Gram stain. Abundant PMNs Intracellular bacteria (potential pathogens) seen on Gram stain. oil field on gs. Intracellular bacteria (potential pathogens) seen on Gram stain. INTERPRETATION: GRAM STAIN: Sputum: 1. Sputum Gram stains should be examined as soon as possible to determine the quality of the specimen. 2. Examine at least 10 fields under low power (10X) to determine the numbers of WBC s and epithelial cell present. 3. IF <25 WBC s / LPF contact the nursing unit or physician and request a new specimen. 4. The physician may elect to have the specimen cultured regardless of the number of WBC s seen on gram stain. 5. Discard plates if the specimen is rejected and report the Gram stain results using the appropriate resulting keypad. Add canned comment under the culture tab Gram stain shows <25 WBC/LPF. Culture not performed. Final verify the order. 6. Reporting the number and morphology of organisms present using the guidelines below. a. Rare# (<1 per oil immersion field) b. Small # (1-5 per oil immersion field) c. Moderate # (5-10 per oil immersion field) d. Large# (>10 per oil immersion field) Specimens other than sputum: 1. Examine Gram stain and report using appropriate resulting keypad. 2. Report the number of WBC s per oil immersion field using the guidelines below. 3. Report the number of morphology of organisms present using the guidelines below. 4. Rare # (<1 per oil immersion field) 5. Small # (1-5 per oil immersion field) 6. Moderate # (5-10 per oil immersion field) 7. Large # (>10 per oil immersion field) GRAM STAIN MORPHOLOGY 1. Positive: Organisms are blue or purple in color. 2. Negative: Organisms are pink or red in color. 3. Rods: Elongated organisms, longer than wide. 4. Cocci: Round organisms, may be in singles, pairs, chains, clusters or tetrads. 5. Yeast: Organisms that may be oval in shape, larger than bacteria, may exhibit budding or pseudohyphae. 6. Coccobacillus: Organisms that appears somewhat elongated, but not enough to call it a rod. INDICATORS OF LOWER RESPIRATORY TRACT PATHOLOGY 1. Inflammatory cells (PMNs) 2. Intracellular bacteria-indicate active phagocytosis Page 3 of 7

4 3. Alveolar macrophages-identified by vacuolated cytoplasm and round eccentric nucleus. 4. Curschmann s spirals (Refer to Figure 2. Page 246 ASM 10 th Ed.) indicate areas within the smear that originated within the alveoli and distal airways. INDICATORS OF ASPIRATION PNEUMONIA Many PMNs and many mixed morph types. Much of the flora is intracellular, and suggestive of strep and anaerobes. CULTURE: 1. Use the gram stain as a guide to interpret the culture. 2. Use the presence of inflammatory cells and bacteria in deciding on the extent of workup. 3. If the culture doesn t match the gram stain, pull the smear and review. 4. Follow Table 1 guidelines for processing and reporting significant organisms. EXAMINE FOR AND ALWAYS REPORT: 1. Streptococcus pyogenes 2. Group B strep in neonates (<3 months) 3. Francisella tularensis 4. Bordetella spp 5. Yersinia pestis 6. Neisseria gonorrhoeae 7. Norcardia 8. Bacillus anthracis 9. Cryptococcus neoformans 10. Molds not considered saprophytic contaminants ALWAYS REPORT, BUT DON T MAKE AN EFFORT TO FIND LOW NUMBERS, UNLESS SEEN IN THE GRAM STAIN. 1. Streptococcus pneumoniae, report sensitivities 2. Haemophilus influenzae, report beta lactamase REPORT IF PRESENT IN SIGNIFICANT AMOUNTS, EVEN IF NOT PREDOMINANT 1. Moraxella catarrhalis 2. Neisseria meningitides Report on inpatients only: IF PRESENT IN SIGNIFICANT AMOUNTS, EVEN IF NOT PREDOMINANT 3. Pseudomonas aeruginosa, report sensitivities 4. Stenotrophomonas maltophilia 5. Acinetobacter, report sensitivities 6. Burkholderia, report sensitivities REPORT IF PRESENT IN SIGNIFICANT AMOUNTS AND THE PREDOMINANT ORGANISM IN THE CULTURE, ESPECIALLY IF THE SMEAR IS SUGGESTIVE OF INFECTION WITH MORPHOLOGY CONSISTENT WITH ISOLATE. 1. Staphylococcus aureus, report sensitivities 2. Single morph type of gram negative rod, report sensitivities Page 4 of 7

5 3. Corynebacterium if urea positive, (C. pseudodiptheriticum) 4. Rhodococcus equi, in immunocompromised patients. REPORT AS NORMAL FLORA LIST SEPARATELY WITH MINIMAL IDENTIFICATION IF 90% PURE CULTURE. 1. Viridans group strep 2. Non-pathogenic Neisseria species 3. Diptheroides 4. Coag negative staph 5. Beta Strep Group F 6. Haemophilus species 7. Eikonella 8. Acinetobacter 9. Capnocytophaga 10. Moraxella species 11. Enterococci 12. Insignificant numbers of Staph aureus,gram negative rods and N. meningitides Alpha strep: Perform a direct bile solubility test or an optochin disc (P disc) on all alpha hemolytic streptococci resembling S. pneumoniae. Refer to Lab-3364 Bile Solubility Test or Lab-3372 Optochin Disc. S. pneumoniae. Alpha strep Bile soluble (dissolves) not bile soluble P disc zone 14mm P disc no zone Beta Strep: Perform a serotyping test (Streptex) Refer to Lab-3367 Test to ID Streptococci. 1. Do only Group A on sputum. Exception: neonates also report Group B beta strep. 2. Do all groups on other lower respiratory cultures. Corynebacterium: Screen for urea positive organism if predominant organism in culture. If Urea (+) identify to rule out C. pseudodiptheriticum. Gamma Strep: 1. Enterococci are considered part of the normal flora of the upper respiratory tract. 2. Do NOT report unless 90% pure culture and identification was by Vitek2 or strep strip. 3. Many gram positive cocci in the normal respiratory tract are PYR, BE and LAP (+) and can be misidentified as enterococci. Refer to Lab-3315 Vitek 2 Identification and Susceptibility Testing or Lab-3301 API Strep. Haemophilus spp: Haemophilus may appear as tiny satelliting colonies on BAP. Comparison of CHOC to BAP should show organisms not readily seen on BAP. Colonies on CHOC agar resembling Haemophilus should be stained. Reisolate any colonies that prove to be lightly stained, pleomorphic gram negative rods to chocolate agar unless well isolated. Test for X and V factor growth requirements, id by VITEK2 or use of API NH Page 5 of 7

6 strip the next day. Refer to Lab-3342 Tests to ID Bacteria Haemophilus X, V, XV Factor Strips, Lab-3315 Vitek 2 Identification and Susceptibility Testing or Lab-3304 API NH Strip. Moraxella and Neisseria spp. (Gram negative diplococcic): Examine chocolate plate for and oxidase (+) colonies that grow poorly on BAP. Confirm ID of N. gonorrhoeae and N.meningitidis. Refer to Lab-3346 Test to Identify Bacteria: Moraxella catarrhalis. Gram negative rods that grow well on MAC: 1. If there is only one morphology of enteric gram negative rods, in significant amounts with no other pathogen in greater amounts, Identify and perform sensitivities. 2. For In-patients, See REPORT IF PRESENT IN SIGNIFICANT AMOUNTS, EVEN IF NOT PREDOMINANT above for additional information. 3. If more than on type of negative rod is present in equal numbers, 4. Enumerate and describe oxidase and indole reactions. 5. Do not perform sensitivities unless physician calls and requests them. Fastidious gram negative rods that grow poorly or not at all on MAC: 1. Perform spot test to differentiate from normal flora. 2. All work on suspicious isolates should be performed under the hood. 3. Refer and isolates that can t be ruled out to Wisconsin State Lab of Hygiene as a possible agent of bioterrorism. Bordetella spp: 1. May grow on BAP after 48 hours 2. Catalase (+) 3. Urea (+) Francisella tularensis: 1. Coccobacili 2. Grow on CHOC but not BAP 3. Does not exhibit satelliting around staph. 4. Weakly catalase (+) 5. Oxidase (+) 6. Urea (+) Legionella: 1. Grow on CHOC but not on BAP 2. Does not exhibit satelliting around staph 3. Appear as circular colonies with entire edges, and exhibit ground glass appearance and iridescent blue, green or pink color. 4. Motility (+) Yesinia pestis: 1. May grow pinpoint on BAP at 24 hours 2. Non-lactose fermenter on MAC Page 6 of 7

7 3. Oxidase (-) 4. Catalase (+) 5. Indole (-) 6. Urea (-) Do not identify other fastidious gram negative rods such as Eikonella unless they are predominant and in large numbers since they are part of normal upper respiratory flora and rarely cause respiratory disease. Staphylococcus spp.: 1. Identify significant amounts of S. aureus using latex agglutination. Refer to Lab-3002 Pastorex Staph Plus. 2. IF Gram stain shows predominant cocci in clusters associated with WBCs and no other pathogen in significant amount, perform sensitivities. Yeast: 1. Yeast will be identified only as Candida albicans using the germ tube test. 2. Refer to Lab-7790 Fungal Identification Yeast. 3. The physician may call for full identification if needed. Mold: Molds are referred to the fungal lab for identification. LIMITATIONS: N/A REVIEW AND CHANGES: This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as the maximum review date. Review will be done by the Technical Leader, Medical Director or designated person. Changes require retyping document or form and review by the Medical Director. REFERENCES: 1. Manual of Clinical Microbiology, Patrick Murray, Ellen Jo Baron, Michael A. Pfaller, Fred Tenover, Robert H. Yolken, ASM, 7 th Edition, ASM Press, Washington D.C Manual of Clinical Microbiology, James Versalovic, Karen C. Carroll, Guido Funke, James H. Jorgensen, Marie Louise Landry and David W. Warnock, ASM 10 th Edition, ASM Press, Washington D.C Specimen Management in Clinical Microbiology, J. Michael Miller, ASM Press, Washington D.C Clinical Microbiology Procedure Handbook, Henry D. Isenberg, 2 nd Edition. Vol 1, ASM Press, Washington D.C Page 7 of 7