1. MICROBIOLOGY IN A HOSPITAL SETTING Kathy Beadle, MHCL MT(ASCP) Microbiology Manager Wesley Medical Center

2. OBJECTIVES Specimen collection: Good and Bad Blood cultures Stool specimens Respiratory specimens Urines Antimicrobial susceptibility testing Gram stains Culture reports

3. You can do that? If you can collect a specimen -- we can culture it! HOWEVER The results are only as good as the specimen obtained.

4. Collecting Quality Specimens Good Specimens –Tissue In large mouth sterile container –Whole fluid In original syringe or container NOT on a swab Bad Specimens –Any specimen collected with a swab –Tissue or fluid placed into a swab tube/device –Any surface specimens

5. FOR QUALITY RESULTS SEND TISSUE AND FLUIDS TO MICROBIOLOGY AS SOON AS POSSIBLE!

6. Swabs don’t do the job… Out of every 100 bacteria absorbed on a swab, only 3 make it to culture. Anaerobes on swabs die upon exposure to air, but survive in tissues and fluids. Swabs hold only 150 microlitres of fluid.

7. Surgical Specimens Rules of Thumb The best specimens are “collected with metal” –Use scalpels, needles and syringes Send fluid in its original container or syringe Collect and send as much specimen as possible Label specimens accurately and completely Reference the anatomical site and describe the specimen

8. TYPES OF BLOOD CULTURES Bacterial –Includes yeast Fungal* –Systemic fungi (Histoplasma, Coccidioides, etc) Mycobacterial* *Requires special collection device

9. OPTIMAL BLOOD CULTURE COLLECTION Prior to starting antibiotics, if possible 2 Separate Venipunctures –Included in order for 1 blood culture –Minimal Time Interval 15 - 20 mL Blood if possible (in adults)

10. Catheter-Related Bloodstream Infection Obtain one culture through line and one by venipuncture –If only one is positive, may be a contaminant Reported with time to positivity or detection –Infected line should become positive at least 2 hours earlier than venipuncture Same organism

11. BLOOD CULTURE WORK-UP Day 0: Culture Drawn Day 1: Positive Culture Detected –Bottle sub-cultured to solid media –Gram-stained smear read and reported –Presumptive tests (if any) set and read

12. BLOOD CULTURE WORK-UP Day 2: Growth on solid media –Identification and Susceptibility tests set –Identification usually complete –Susceptibility test may be complete Day 3: Susceptibility test usually complete

13. BLOOD CULTURES Cultures held 5 days before being finaled as “No growth” Most “fastidious” organisms detected within routine incubation time –Franciscella tularensis –Aggretibacter –Cardiobacterium hominis

14. STOOL SPECIMENS Routine Culture –Salmonella –Shigella –Campylobacter –Shiga-Toxin producing E coli (not just O157) Notify the laboratory if you suspect an unusual pathogen

15. Ova and Parasite Exam 1 - 3 Specimens (only 1 per day!) Consider ordering specific tests for Giardia/Cryptosporidium Inpatients: < 72 hr since admission

16. Clostridium difficile-Associated Diarrhea Symptoms include fever, abdominal cramping and diarrhea –Formed specimens rejected Notify the lab if toxic megacolon suspected 1 Specimen Usually Sufficient “Community-acquired” infections becoming more common

17. Clostridium difficile General rule: If the stick stands, the test is banned.

18. Respiratory Specimens for Bacterial Culture Tracheal Aspirate Sputum –Evaluated by gram stain for adequacy Bronchial Alveolar Lavage (BAL)

19. Respiratory Specimens for Bacterial Culture Mini-BAL –Patient on ventilator –Obtained by RT using special catheter –Cultured quantitatively to guide interpretation Potential pathogens present in >10,000 col/mL reported

20. Respiratory Specimens for Virus Best specimen: Nasopharyngeal aspirate 2 nd Best: Nasopharyngeal swab Rapid assays: –Restricted offering –These tests are not the best for diagnosis –A negative result does not mean the patient does not have influenza or RSV

21. Respiratory Specimens for Pertussis Nasopharyngeal specimens only PCR is performed daily on 1 st shift at VC –Specimens from Wesley are sent to VC

22. Urine Cultures Specimens –Clean-catch (voided) –Urinary Catheter Culture Work-up –Reflex Cultures VC: Urinalysis specimens that contain >5 WBC WMC: Urinalysis specimens that contain >20 WBC –Single organism at >10,000 colonies/mL

23. Urine for Legionella antigen Tests are run throughout the day and night Legionella antigen test is only for Serogroup 1 –Only 70% of Legionella infections are Serogroup 1

24. Streptococcus pneumonia antigen Specimen type may be either urine or CSF Urine for Streptococcus pneumonia antigen may give a false positive if the patient has been vaccinated within 5 days prior for pneumococcus. – WMC: A comment will appear on positive Strep pneumo antigens

25. ANTIMICROBIAL SUSCEPTIBILITY TESTING Synonyms: –AST = Antimicrobial susceptibility testing –Sensitivity MIC = Minimum inhibitory concentration Vitek = automated method E Test = manual method Microscan = manual method Kirby Bauer = manual method

26. VITEK 2 E-TEST KIRBY BAUERMICROSCAN PLATE

27. ANTIMICROBIAL SUSCEPTIBILITY TESTING Standardized Tests Defined by Clinical and Laboratory Standards Institute –Bacteria –Fungi –Mycobacteria Research Procedures or Not Available: –Viruses –Parasites

28. ANTIMICROBIAL SUSCEPTIBILITY TESTING Qualitative: (Kirby Bauer) –Disk Diffusion (S, I, or R) Quantitative: (Vitek, E test, Microscan) –Minimum Inhibitory Concentration (MIC)

29. Antimicrobial Susceptibility Reporting MIC value –Based on 2-fold dilution of antibiotic –Antibiotic with lowest number not necessarily best

30. Antimicrobial Susceptibility Reporting Interpretation –S, I or R –Based on achievable levels of antibiotic Does not consider concentration of antibiotics in urine or other body fluids Does not consider penetration into tissues or cells

31. AST NOT ROUTINELY PERFORMED Bacteria that are rarely significant Bacillus species Corynebacteria Gardnerella vaginalis Lactobacillus species

32. AST NOT ROUTINELY PERFORMED Bacteria with Predictable Susceptibility Patterns Except from Blood or CSF cultures –Groups A and B Streptococci –Haemophilus species β-lactamase tested and reported routinely –Stenotrophomonas maltophilia Trimethoprim/sulfamethoxazole usually used –Moraxella

33. Exception: Group B Strep at WMC Patient with a listed penicillin allergy and growth of Group B Strep (GBS): –MIC is set and reported –D-Test for inducible clindamycin resistance is set at the same time –Clindamycin will only be reported as susceptible if there is no inducible resistance

34. D Test to Detect Clindamycin Resistance

35. AST NOT ROUTINELY PERFORMED Bacteria Requiring Special or Nonstandard Testing Conditions Fastidious Gram Negative Rods –eg. Eikenella, Campylobacter Anaerobes from Mixed Cultures Anaerobes usually susceptible to: Beta-Lactam/Inhibitor Combinations Metronidazole Carbapenems

36. Additional Resistance Screening Enteric Gram negative bacteria –ESBL: extended-spectrum β lactamase Resistant against all β-Lactam antibiotics Streptococci and Staphylococci –D test for Clindamycin resistance where appropriate

37. Screens for Antimicrobial Resistance Streptococcus pneumoniae: Penicillin Enterococcus species: Vancomycin Staphylococcus aureus: Oxacillin

38. Streptococcus pneumoniae Ceftriaxone Interpretation Separate breakpoints based on site of infection –Meningitis Based on achievable CSF levels S: 2 mcg/mL –Nonmeningitis Based on achievable serum levels S: 4 mcg/mL

39. Streptococcus pneumoniae Penicillin Interpretation Separate breakpoints based on site of infection At this time the breakpoint interpretations for penicillin are reported below the MIC – Non-meningitis pneumococcal isolates with a penicillin MIC <=0.06 can be considered to be sensitive to oral penicillins – Non-meningitis pneumococcal isolates with penicillin MIC 0.12 - 2.0 can be considered to be sensitive to IV penicillin or oral ampicillin – Pneumococcal Meningitis should not be treated with penicillin unless the MIC <=0.06

40. Antibiotics and Susceptibility Testing Don’t hesitate to contact the Microbiology Laboratory with questions –Best time to call: 1 st Shift (7am-2:30pm)! –Appropriate antibiotics usually reported –Do not ask the Laboratory to recommend an antibiotic to treat a specific patient Contact the Pharmacy for questions about dosing and pharmacology

41. GRAM STAINS Gram stains are preliminary tests What we see may not grow, and what grows we may not see on the gram stain Gram positive cocci resembling staphylococcus What we cannot tell you from the gram stain: Staph aureus vs Staph epi Gram positive cocci resembling streptococcus Sometimes we can tell you if it looks like Strep pneumo W e cannot tell you if it is enterococcus

42. GRAM STAINS Gram negative rods: –Sometimes we can tell if it looks like an enteric, pseudomonas, or Haemophilus –What we cannot tell you is which enteric gram negative rod. (E.coli, Kleb, Proteus, etc)

43. CULTURE REPORTS First day of growth of Staph aureus –We cannot tell you if it is Methicillin Resistant Staph aureus. (MRSA) First day of growth of gram negative bacilli –We cannot tell you the organism name-but we might be able to give you a good idea. Just remember, we might be wrong.

44. CULTURE REPORTS Streptococcus on plate media may be alpha, beta, or gamma in appearance. Alpha strep: –Streptococcus pneumonia –Streptococcus viridans –Enterococcus

45. CULTURE REPORTS Beta strep: Groups A,B,C,D,F,G and non- groupable. –Group A = Streptococus pyogenes –Group B = Streptococcus agalactiae –Group D = Enterococcus

46. CULTURE REPORTS Gamma strep: Streptococcus viridans or enterococcus –Streptococcus viridans: frequently part of the normal body flora, rarely a pathogen –Enterococcus: species, faecalis, or faecium Enterococcus antibiotic screen –If sensitive to gentamicin, vancomycin, and ampicillin no further ID/MIC is done –Exception: blood cultures; CSF

47. CULTURE REPORTS Sterile sites: blood, tissue, body fluid –Organisms from these sites will be considered likely pathogens Non-sterile sites: gastrointestinal tract, respiratory tract –Organisms from these sites will be evaluated for normal flora and pathogenic flora

48. CULTURE REPORTS Microbiology is not a CSI television show –There is no piece of equipment we can shoot the specimen into and get an answer within 45 minutes Bacteria have their own timetable for growth and some have special nutritional needs or restrictions Sometimes an MIC takes more than one try and more than one method to get an answer

49. CULTURE REPORTS Cultures that are reported as No Growth will NOT have a sensitivity Non-approved or Non-validated testing will most likely be rejected Write clear and concise orders for your culture specimens and sign your name –If we have a question we know who to page

50. CULTURE REPORTS We call critical results to the nurse taking care of the patient and they notify the physician We will tell you as much as we can as soon as we know

51. WE ARE JUST A PHONE CALL AWAY Don’t hesitate to contact the Microbiology Laboratory with questions –Best time to call: 1 st Shift (7am-2:30pm) –Best time to visit:1 st Shift (7am-2:30pm)