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MICROBIOLOGY IN A HOSPITAL SETTING Kathy Beadle, MHCL MT(ASCP) Microbiology Manager Wesley Medical Center OBJECTIVES • • • • • • • • Specimen collection: Good and Bad Blood cultures Stool specimens Respiratory specimens Urines Antimicrobial susceptibility testing Gram stains Culture reports 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. 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 FOR QUALITY RESULTS SEND TISSUE AND FLUIDS TO MICROBIOLOGY AS SOON AS POSSIBLE! 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. 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 TYPES OF BLOOD CULTURES • Bacterial – Includes yeast • Fungal* – Systemic fungi (Histoplasma, Coccidioides, etc) • Mycobacterial* *Requires special collection device 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) 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 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 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 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 STOOL SPECIMENS • Routine Culture – Salmonella – Shigella – Campylobacter – Shiga-Toxin producing E coli (not just O157) • Notify the laboratory if you suspect an unusual pathogen Ova and Parasite Exam • 1 - 3 Specimens (only 1 per day!) • Consider ordering specific tests for Giardia/Cryptosporidium • Inpatients: < 72 hr since admission 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 Clostridium difficile • General rule: If the stick stands, the test is banned. Respiratory Specimens for Bacterial Culture • Tracheal Aspirate • Sputum – Evaluated by gram stain for adequacy • Bronchial Alveolar Lavage (BAL) 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 Respiratory Specimens for Virus • Best specimen: Nasopharyngeal aspirate • 2nd 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 Respiratory Specimens for Pertussis • Nasopharyngeal specimens only • PCR is performed daily on 1st shift at VC – Specimens from Wesley are sent to VC 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 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 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 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 VITEK 2 KIRBY BAUER E-TEST MICROSCAN PLATE ANTIMICROBIAL SUSCEPTIBILITY TESTING • Standardized Tests Defined by Clinical and Laboratory Standards Institute – Bacteria – Fungi – Mycobacteria • Research Procedures or Not Available: – Viruses – Parasites ANTIMICROBIAL SUSCEPTIBILITY TESTING • Qualitative: (Kirby Bauer) – Disk Diffusion (S, I, or R) • Quantitative: (Vitek, E test, Microscan) – Minimum Inhibitory Concentration (MIC) Antimicrobial Susceptibility Reporting • MIC value – Based on 2-fold dilution of antibiotic – Antibiotic with lowest number not necessarily best 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 AST NOT ROUTINELY PERFORMED • Bacteria that are rarely significant • • • • Bacillus species Corynebacteria Gardnerella vaginalis Lactobacillus species 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 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 D Test to Detect Clindamycin Resistance 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 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 Screens for Antimicrobial Resistance • Streptococcus pneumoniae: Penicillin • Enterococcus species: Vancomycin • Staphylococcus aureus: Oxacillin Streptococcus pneumoniae Ceftriaxone Interpretation • Separate breakpoints based on site of infection – Meningitis • Based on achievable CSF levels • S: < 0.5 I: 1 R: > 2 mcg/mL – Nonmeningitis • Based on achievable serum levels • S: < 1 I: 2 R: > 4 mcg/mL 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 Antibiotics and Susceptibility Testing • Don’t hesitate to contact the Microbiology Laboratory with questions – Best time to call: 1st 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 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 • We cannot tell you if it is enterococcus 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) 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. CULTURE REPORTS • Streptococcus on plate media may be alpha, beta, or gamma in appearance. • Alpha strep: – Streptococcus pneumonia – Streptococcus viridans – Enterococcus CULTURE REPORTS • Beta strep: Groups A,B,C,D,F,G and nongroupable. – Group A = Streptococus pyogenes – Group B = Streptococcus agalactiae – Group D = Enterococcus 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 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 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 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 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 WE ARE JUST A PHONE CALL AWAY • Don’t hesitate to contact the Microbiology Laboratory with questions – Best time to call: 1st Shift (7am-2:30pm) – Best time to visit:1st Shift (7am-2:30pm)