Respiratory Cultures. Section Four: CLS419 Clinical Microbiology II University of Nebraska Medical Center Clinical Laboratory Science Program

Transcription

1 CLS419 Clinical Microbiology II University of Nebraska Medical Center Clinical Laboratory Science Program Section Four: Respiratory Cultures CLS 419 Clinical Microbiology II Page 62

2 Respiratory Cultures Task List 1. Review objectives for this unit Objectives for all CLS 419 Bacteriology Units Rotation II Respiratory Cultures II 2. Read: Textbook: Chapter 26 Mycobacterium Tuberculosis and Other Non-Tuberculous Mycobacteria, pages Chapter 32 Upper and Lower Respiratory Tract Infections, pages Chapter 19 B Legionella, pages Chapter 19 C Bordetella, pages Procedures: Respiratory Cultures Procedure For Rotations (found in Rotation I Manual) Group A Streptococcus Screening Procedure (found in Rotation I Manual) Respiratory Cultures Special Requests (found in Rotation I Manual) 3. Review: Supplemental Lecture Information: Fastidious Gram-Negative Bacilli Respiratory Cultures PowerPoint (from Rotation I) 4. Complete Mycobacteria Online Module a. View Mycobacteria PowerPoints: Part I: Safety and General Characteristics Part II: Specimen Collection Part III: Specimen Processing Part IV: Microscopic Examination Part V: Culture Media and Isolation Part VI: Identification and Susceptibility Testing Part VII: Mycobacteria tuberculosis complex Part VIII: Mycobacteria other than M. tuberculosis complex b. View CDC training presentation: TB Fluorochrome Staining (optional) c. View videos / animations: Mycobacteria Processing: Safety Mycobacteria Culture Specimen Processing Part I, Part II and Part III Mycobacteria Culture Incubation Acid Fast Staining Using Ziehl-Neelsen Method d. Complete National Tuberculosis Curriculum Consortium Computerized Cases: Percy Edwards for CLS (Y4) Students (required) e. Complete the mycobacteria study questions f. Complete the online Mycobacteria Quiz (graded) Note: This online quiz must be taken prior to the Respiratory II Exam. 5. Complete Respiratory Cultures II Study Questions 6. Complete bench time at your clinical site as assigned by your instructor(s) 7. Complete Respiratory II Exam CLS 419 Clinical Microbiology II Respiratory Cultures Page 63 Task List and Objectives

3 RESPIRATORY CULTURES NOTE: All objectives in bold face are new objectives for CLS 419 and were not part of CLS 418. Objectives: After review of the Clinical Microbiology Study Manual and completion of the clinical rotation for CLS 419 Clinical Laboratory Microbiology II, the Clinical Laboratory Science student will be able to: 1. Describe the proper method for collecting each of the following types of cultures: a. Throat b. Nasopharyngeal c. Sputum 2. Evaluate the suitability of a respiratory specimen for culture using a direct specimen Gram stain. 3. Describe the procedures for setting up a culture on the following respiratory specimens, including the types of media used and incubation requirements: a. Throat b. Sputum c. Tracheal aspirate d. Transtracheal aspirate e. Bronchial washing/bronchial alveolar lavage/bronchial brushings f. Sputum from a patient with cystic fibrosis 4. Correlate direct specimen Gram stain results with respiratory culture interpretations. 5. Evaluate the normal flora of the following respiratory sites: a. Upper respiratory tract b. Lower respiratory tract 6. Evaluate pathogens commonly found in the following respiratory sites: a. Upper respiratory tract b. Lower respiratory tract c. Respiratory tract of patients with cystic fibrosis 7. Compare the advantages and disadvantages of molecular-based tests with other laboratory test methods utilized in these clinical applications: a. Detection of Bordetella pertussis b. Rapid identification of Mycobacterium species MYCOBACTERIACEAE 1. Define the following terms in relation to mycobacteria: a. Acid-fast b. Mucolytic agent c. Digestion d. Decontamination 2. Describe the general microbiologic characteristics of mycobacteria, including: a. Transmission b. Cell structure c. Growth characteristics d. How these characteristics are used in laboratory identification 3. Describe biosafety precautions necessary when collecting, handling, transporting and processing specimens for mycobacterial culture. CLS 419 Clinical Microbiology II Respiratory Cultures Page 64 Task List and Objectives

4 4. Explain the following pre-analytical issues for specimens of suspected mycobacteria infection: a. Specimen collection 1) Sputum cultures 2) Blood cultures 3) Urine cultures 4) Other specimens b. Specimen transport 1) In house laboratory 2) Reference laboratory 5. Describe appropriate digestion and decontamination procedures on specimens submitted for mycobacterial culture, including the purpose of each reagent. 6. Describe the principle, indications, advantages, limitations, sensitivity and specificity of the following tests used in the diagnosis of mycobacterial infections: a. Tuberculosis screening tests 1) in vivo (TB skin test/ PPD/Mantoux) 2) in vitro (Quantiferon) b. Acid-fast stain 1) Carbolfuchsin methods 2) Fluorochrome methods c. Acid-fast culture 1) Liquid-based media (Bactec) 2) Solid-based media (Lowenstein-Jensen, Middlebrook) d. Identification methods 1) Biochemical 2) Molecular e. Susceptibility tests 7. Interpret acid-fast smears. 8. Describe the characteristic colony morphology of Mycobacterium tuberculosis on solid-based media. 9. Explain the purpose of performing a conventional niacin test. 10. Correlate the physical symptoms, pathophysiology, and clinical laboratory findings, including growth rate, incubation temperature and photoreactivity, with the following mycobacterial species: a. Mycobacterium tuberculosis b. Mycobacterium avium complex (MAC) c. Mycobacterium kansasii d. Mycobacterium fortuitum-chelonei complex e. Mycobacterium marinum 11. Discuss the clinical significance of the following mycobacterial species: a. Mycobacterium bovis b. Mycobacterium scrofulaceum c. Mycobacterium gordonae d. Mycobacterium leprae 12. Correlate acid-fast smear findings to culture results from a patient with Mycobacterium leprae. CLS 419 Clinical Microbiology II Respiratory Cultures Page 65 Task List and Objectives

5 AEROBIC GRAM-NEGATIVE COCCI 1. Describe the following characteristics common to the Neisseriaceae: a. Gram stain morphology b. Oxidase production 4. Explain the principle of the following biochemical reactions and other characteristics, including the interpretation of results: a. Oxidase production d. Growth on selective media b. DNase production e. Growth on simple media (i.e. Nutrient agar) c. Nitrate reduction f. Butyrate esterase 5. Evaluate the acceptability of results for tests listed in Objective #4, including appropriate corrective action and/or further testing if necessary. 6. Differentiate between the following organisms based on the reactions listed in objective #4, as well as carbohydrate utilization (glucose, maltose, lactose, sucrose): c. Neisseria species d. Moraxella (Branhamella) catarrhalis 8. Correlate the clinical, epidemiological and laboratory findings associated with the following infections: c. Moraxella (Branhamella) catarrhalis 1) Otitis media/maxillary sinusitis in children 2) Pneumonia/bronchitis in immunocompromised HAEMOPHILUS 1. Describe the specific specimen processing requirements for Haemophilus species, including special media (nutritional requirements) and incubation times. 2. Describe the characteristic Gram stain and colonial morphology of Haemophilus species. 3. Explain the principle of the following biochemical reactions and other characteristics, including the interpretation of results: a. X and V requirements c. Hemolysis on horse blood agar b. Porphyrin test d. Satellitism 4. Evaluate the acceptability of results for tests listed in Objective #3, including appropriate corrective action and/or further testing if necessary. 5. Differentiate between the following species of Haemophilus, based on the tests listed in Objective #3. a. Haemophilus influenzae b. Haemophilus parainfluenzae 6. Describe the significance of the capsule as a virulence factor. 8. Correlate the clinical, epidemiological and laboratory findings associated with the following infections: a. Haemophilus influenzae 1) Respiratory infections 9. Discuss the following antimicrobial tests for Haemophilus influenzae: a. Beta-lactamase testing b. Disk diffusion c. Microtiter dilution CLS 419 Clinical Microbiology II Respiratory Cultures Page 66 Task List and Objectives

6 FASTIDIOUS GRAM-NEGATIVE RODS 1. Define the term fastidious, as used in clinical microbiology. 2. Discuss appropriate specimen collection for the isolation of Bordetella pertussis. 3. Evaluate the following media, including purpose, proper use, inhibitory or selective properties and colonial appearance: a. Blood cysteine c. Bordet-Gengou b. Regan-Lowe d. Buffered charcoal yeast extract (BCYE) 4. Describe specific specimen processing requirements, including special media, incubation times and any characteristic colonial morphology for each of the following: b. Francisella species c. Bordetella species d. Legionella species 6. Discuss the following techniques used to enhance Gram stain morphology: a. Extended safranin b. Substitution of carbolfuschin 7. Assess the use of immunologic methods for direct specimen detection and/or organism identification of the following organisms: a. Francisella tularensis b. Bordetella pertussis d. Legionella species 10. Correlate the clinical, epidemiological and laboratory findings associated with the following infections: a. Francisella tularensis 1) Tularemia b. Bordetella pertussis 1) Pertussis (whooping cough) d. Legionella species 1) Legionnaire s disease 2) Pontiac fever 11. Evaluate the appropriateness of susceptibility testing of the organisms listed in objective #4 GLUCOSE NONFERMENTING GRAM-NEGATIVE RODS 2. Distinguish the following glucose nonfermenting gram-negative rods from Enterobacteriaceae, based on glucose utilization and cytochrome oxidase production: a. Pseudomonas aeruginosa b. Acinetobacter species c. Stenotrophomonas maltophilia f. Burkholderia cepacia 3. Differentiate the glucose nonfermenting gram-negative rods listed in Objective #2, using the following characteristics: a. Glucose fermentation d. Polymyxin susceptibility b. Growth on MacConkey agar e. Gram stain reaction c. Cytochrome oxidase 5. Evaluate the role of Pseudomonas aeruginosa in the following clinical conditions: a. Nosocomial infections c. Respiratory infections g. Cystic fibrosis CLS 419 Clinical Microbiology II Respiratory Cultures Page 67 Task List and Objectives

7 7. Evaluate Pseudomonas aeruginosa antimicrobial susceptibility results based upon established predictability patterns to the antimicrobial agents listed: a. Ampicillin b. 1st & 2nd generation cephalosporins GRAM-POSITIVE BACILLI 1. Describe the Gram stain morphology, oxygen requirements and catalase reaction for each of the following gram-positive bacilli: b. Irregularly shaped, non-spore-forming 1) Corynebacterium species 2. Describe specimen collection and processing for the isolation of Corynebacterium diphtheriae. 4. Evaluate the following media, including purpose, proper use, significant ingredients and colonial appearance: a. Loeffler s media b. Tellurite media (Tinsdale) 5. Interpret the following test results used to identify gram-positive bacilli: a. Urease b. Nitrate reduction 6. Evaluate the acceptability of results for tests listed in Objective #5, including appropriate corrective action and/or further testing if necessary. 7. Identify the following aerobic gram-positive bacilli based on the Gram stain, colony morphology and carbohydrate utilization, as well as the tests listed in Objective #5: c. Corynebacterium species/diphtheroids 9. Correlate the clinical, epidemiological and laboratory findings associated with the following infections: c. Corynebacterium diphtheriae 1) Diphtheria 10. Describe the use of the in vivo and in vitro methods of determining toxigenicity of Corynebacterium diphtheriae. 11. Correlate the patient immune status with the pathogenicity of the following organisms: a. Corynebacterium jekeium 12. Describe the typical antibiotic susceptibility patterns of Corynebacterium jekeium. STAPHYLOCOCCUS 4. Correlate clinical, epidemiological and laboratory findings associated with the following infections: a. Staphylococcus aureus 5) Pneumonia STREPTOCOCCACEAE 3. Discuss the clinical symptoms and epidemiology associated with the following infections: a. Group A Streptococcus (Streptococcus pyogenes) 1) Pharyngitis b. Group B Streptococcus (Streptococcus agalactiae) 1) Neonatal pneumonia f. Streptococcus pneumoniae 1) Pneumonia 4) Sinusitis CLS 419 Clinical Microbiology II Respiratory Cultures Page 68 Task List and Objectives

8 4. Correlate the clinical and laboratory findings associated with the following post-streptococcal sequelae: a. Acute glomerulonephritis b. Rheumatic fever ENTEROBACTERIACEAE 9. Correlate the clinical, epidemiological and laboratory findings associated with Enterobacteriaceae related to the following infections: f. Opportunistic/nosocomial infections g. Respiratory infections 10. Evaluate susceptibility results for the following organisms based on established predictable patterns to the antimicrobial agents listed: a. Klebsiella pneumoniae to ampicillin CLS 419 Clinical Microbiology II Respiratory Cultures Page 69 Task List and Objectives

9 CLS 419 Mycobacteria Study Worksheet Questions 1. Define the term acid fast. 2. Define the term mucolytic agent. 3. Define the term digestion. 4. Define the term decontamination. 5. True or false. Morphologically, mycobacteria are rod shaped bacteria which appear beaded or banded when stain. 6. True or false. Mycobacteria are aerobic, non-spore forming gram positive rods that require no special growth requirements. 7. Describe the special safety precautions necessary in the mycobacteria laboratory. 8. Briefly describe the digestion/decontamination/concentration procedure used for sputum samples when culturing for mycobacteria. CLS 419 Clinical Microbiology II Respiratory Cultures Page 70 Mycobacteria Study Questions

10 9. When setting up a sputum sample for mycobacteria, what media should be used for optimal recovery of mycobacteria? 10 Evaluate the appropriate use of the Ziehl-Neelsen and Auramine O stain. Include methodology, specificity and sensitivity, and advantages and disadvantages. 11. Explain the terms typical and atypical mycobacteria. 12. True or false. Mycobacterium kansasii is distinguishable from Mycobacteria tuberculosis based on patient symptoms. 13. What is the causative agent of leprosy? 14. Describe the typical acid fast stain of Mycobacteria tuberculosis? 15. Which Mycobacterium sp. can cause skin infections and grows best at 30 degrees Celsius? a. Mycobacterium kansasii b. Mycobacterium tuberculosis c. Mycobacterium marinum d. None of the above 16. Which Mycobacterium sp. is found primarily in surgical wounds or trauma? 17. Which Mycobacterium sp. is a common tap water contaminate? 18. What biochemical test is used to identify Mycobacteria tuberculosis? CLS 419 Clinical Microbiology II Respiratory Cultures Page 71 Mycobacteria Study Questions

11 Study Questions Respiratory Cultures Rotation II Utilize your Respiratory Culture Rotation II objectives as a guide for answering the following questions. Suggested references in completing the study questions: Mahon & Manuselis, Textbook of Diagnostic Microbiology, 3 rd Ed., Saunders, (Your textbook) Forbes, B.A., Sahm, D. F., & Weissfeld, A.S., Bailey and Scott s Diagnostic Microbiology, 11 th Ed, Mosby, Koneman, Color Atlas and Textbook of Diagnostic Microbiology, 5 th Ed., J.B. Lippincott Co., (available at each site) Student Manuals When indicated: Clinical Microbiology Blackboard course components are found under the <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions> Folder. 1. A 78-year-old male in AICU tracheal aspirate s direct specimen Gram stain was reported as: Moderate WBCs Moderate gram-negative rods After 18 hours of incubation at 35 C, the culture is growing: 5% CO 2 : BAP- many mucoid, gray colonies CHOC many mucoid, gray colonies Ambient air: MAC many clear colonies What is your next step? After recording your answer, go to the Clinical Microbiology Blackboard course component <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions><Question 1> to continue with this question. Record further results here: CLS 419 Clinical Microbiology II Respiratory Cultures Page 72 Study Questions

12 2. A 17-year-old male cystic fibrosis patient s sputum direct specimen Gram stain was reported as: Moderate WBCs Moderate gram-negative rods After 18 hours of incubation at 35 C, the culture is growing: 5% CO 2 : BAP- many tiny, yellowish colonies CHOC many tiny, yellowish colonies Ambient air: MAC many tiny clear colonies What is your next step? After recording your answer, go to the Clinical Microbiology Blackboard course component <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions><Question 2> to continue with this question. Record further results here: CLS 419 Clinical Microbiology II Respiratory Cultures Page 73 Study Questions

13 3. A four-month-old female in PICU tracheal aspirate s direct specimen Gram stain was reported as: Many PMNs Many gram-negative rods After 18 hours of incubation at 35 C, the culture is growing: 5% CO 2 : BAP- many large mucoid, gray colonies CHOC many large, gray colonies Ambient air: MAC many mucoid, light pink colonies What is(are) your next step(s)? After recording your answer, go to the Clinical Microbiology Blackboard course component <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions><Question 3> to continue with this question. Record further results here: Which of the following susceptibility patterns do you expect with this organism? A. Ampicillin resistance B. Gentamicin resistance C. Penicillin susceptibility D. Vancomycin susceptibility Record your answer and continue to page 4 of this learning unit in the Blackboard course component. CLS 419 Clinical Microbiology II Respiratory Cultures Page 74 Study Questions

14 4. A throat swab from an unvaccinated 4-year-old male is submitted for a diphtheria culture. The culture is set up on routine sheep blood agar (5% CO 2 ) and cystine-tellurite blood agar (ambient air). After 48 hours of incubation, moderate small, black colonies grew on the tellurite agar. One of the small black colonies was subcultured to a routine BAP. Go to the Clinical Microbiology Blackboard course component <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions><Question 4> to view the Gram stain result of the subcultured organism. Subculture Gram stain result: Record further results here: 5. A throat swab from a 2-year-old is received for a Group A Strep Screen. The rapid screening test was negative. After 18 hours of incubation at 35 in 5% CO 2, rare suspicious colonies were seen. This colony was subcultured to a sheep blood agar plate. Go to the Clinical Microbiology Blackboard course component <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions><Question 5> to view the colony morphology of the subcultured organism. Record further results here: 6. Rheumatic fever and glomerular nephritis occur in patients because: a. Streptococcus pneumonia infection was not treated early. b. Early treatment of Group A Streptococcus was not administered. c. The causative organism quickly invades the central nervous system. d. The organism will quickly invade the lower respiratory tract causing a necrotizing pneumonia 7. Francisella tularensis requires what to be added to media in order to grow? 8. A sputum culture from a 10-year-old cystic fibrosis patient grew gram-negative coccobacillus on both the blood agar plate and MacConkey (clear colonies). Pseudomonas aeruginosa is suspected. What findings would you suspect for the following reactions? Oxidase: Glucose tube with oil overlay: Glucose tube without oil overlay: CLS 419 Clinical Microbiology II Respiratory Cultures Page 75 Study Questions

15 9. A 32-year-old male is admitted into the hospital complaining of chronic cough lasting for past several weeks. The patient also complains of night sweats and lost of appetite. The patient has recently immigrated into the United States. The initial gram stain of a sputum sample shows many grampositive rods with a beaded appearance? Which of the following test would be you suggest the physician order next? a. Blood cultures b. Urine Cultures c. Acid-fast stain and culture d. Fungal culture Which organism do you expect and what are your expect results? Why is it important to diagnosis this infectious agent? 10. The purpose of the Na citrate in the N-acetyl-L-cysteine digestion and decontamination procedure for acid-fast bacteria is: a. Decontaminate the specimen of non-acid-acid fast bacteria b. To serve as a mucolytic agent to liquefy mucous c. Stabilize the acetyl-cysteine by binding metal ions. d. Stop the decontamination/digestion process so that acid-fast bacilli are not killed. e. Both C and D. f. All the above Explain your answer. CLS 419 Clinical Microbiology II Respiratory Cultures Page 76 Study Questions

16 11. The following results were obtained from a 6-month-old child s sinus culture: Specimen gram stain: Many WBC s Many gram-negative coccobacilli Many encapsulated gram-positive cocci in pairs and chains Many gram-negative diplococci BAP: Isolate #1: Many alpha-hemolytic colonies with a checker-like appearance Isolate #2: Many grayish white granular colonies CHOC: Isolate #1: Many clear colonies with a yellowing of the agar Isolate #2: Many grayish-white granular colonies Isolate #3: Many grayish colonies MAC: No growth For isolate #1 which of the following would be needed to confirm identification? a. Oxidase b. Optochin susceptibility c. Bile Solubility d. Both B and C. e. None of the above What is your suspected pathogen for isolate # 1? Before finalizing the culture report, what must be done with respect to antimicrobial testing on this isolate? For isolate #2 what is your probable organism? For isolate #2 list the test needed to confirm identification with appropriate result(s). In this specimen is isolate #2 considered a pathogen? Explain. For isolate #3 what organism is suspected? Is isolate #3 considered a pathogen in this specimen? Why or why not? CLS 419 Clinical Microbiology II Respiratory Cultures Page 77 Study Questions

17 12. A digested/decontaminated/concentrated sputum specimen has the following results on an Auramine fluorochrome stain: Yellowish-orange rods against a black background A Ziehl-Neelsen stain was performed on the slide over the Auramine fluorochrome stain. Go to the Clinical Microbiology Blackboard course component <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions><Question 12> to see the Ziehl-Neelsen stain result. These results are most consistent with: a. Legionella species b. Mycobacterium fortuitum c. Mycobacterium species d. Nocardia species Explain your answer: 13. An AFB sputum culture s automated broth medium is positive at 10 days. A Ziehl-Neelsen stain performed on the broth is positive. The egg-based agar tube incubated at 37C showed buff-colored growth at 15 days. Go to the Clinical Microbiology Blackboard course component <Assignments><Clinical Rotations - Bacteriology><Respiratory Culture II Unit><Study Questions><Question 13> for further identification information. a. Record biochemical/identification test results for: 1. Pigmentation production when exposed to light: 2. Niacin test: 3. Nitrate reduction: b. This is most consistent with what organism? CLS 419 Clinical Microbiology II Respiratory Cultures Page 78 Study Questions