Microbiology Basics: Collaborating with Infection Prevention and Control

Transcription

1 Microbiology Basics: Collaborating with Infection Prevention and Control Dr. Heather Adam, PhD, FCCM, D(ABMM) Clinical Microbiologist, Diagnostic Services of Manitoba June 14, 2013

2 Objectives Review of basic microbiology Discuss the importance of specimen collection Review of interpretation of microbiology results The importance of a collaborative relationship between infection prevention and control and the laboratory

3 REVIEW OF BASIC MICROBIOLOGY

4 Microorganism Categorization Classification of microorganisms Kingdom Division Class Order Family Genus Species Bacteria: Single-celled microorganisms which can exist either as independent (free-living) organisms or as parasites Virus: Ultramicroscopic infectious, obligate intracellular parasite with a genome composed of RNA or DNA Fungi: Multicellular organisms, includes yeast and moulds

5 Bacterial Genetics For all organisms, hereditary information is encoded in nucleic acids DNA: deoxyribonucleic acid RNA: ribonucleic acid Order of bases along DNA or RNA strand = base sequence i.e. genetic code DNA sequence encodes a product = gene All genes in an organism = genome Genome is organized into discrete elements = chromosomes Bacteria have 1 chromosome/cell Fungi & parasite have >1 chromosome/cell Viruses genome can be DNA or RNA

6 Bacterial Genetics con t Plasmids = mini chromosomes Variable #/bacterial cell Encode genes for replication, transfer between bacterial cells, survival advantages (eg. antibiotic resistance genes) but not products for viability May become incorporated in the chromosome Transposable elements DNA that move from one genetic element to another ie. Plasmid to chromosome or vice versa Insertion sequences & transposons

7 Microbiological Testing - Microscopy Light microscopy Gram stain (bacteria), Kinyoun/Zeil-Neilson (TB and AFB), India ink (cryptococus), KOH (fungal elements), Trichrome (stool parasites), Giemsa (blood parasites), wet preps (stool parasites, vaginal swabs - Trichomonas), Iodine (stool parasites). Fluorescent microscopy Calcofluor (fungi) DFA (viruses and others), Acridine (DNA), Auramine (TB) Darkfield Syphilis (historical)

8 The Gram Stain In the late 1800 s, Christian Gram observed that some bacteria retained a dye-iodine complex when rinsed with alcohol, while other genera were easily decolorized with alcohol and could be then visualized by a contrasting counterstain. This staining procedure defines two bacterial groups: Those which retain the primary dyes ( Positive by Gram s Method or Gram-Positive ) Those which are easily decolorized ( Negative by Gram s Method or Gram-Negative ). This is the starting point for bacterial identification procedures.

9 Gram- Negative bacteria have thin walls surrounded by lipid-rich membranes Gram- Positive bacteria have thick, dense, relatively nonporous walls

10 Examples of Gram Stains Gram positive Gram negative Cocci Bacilli

11 Other Stains Calcofluor: Blastomyces Auramine: TB Dept. Micro., Mt. Sinai Hosp. Wet Mount: Trichomonas Kinyoun: TB Univ. Washington

12 Microbiological Testing - Culture Most organisms can be cultured, but it s not always simple Bacteria Most require nutrients, humidity and heat. Some need special media or atmospheres. Majority grow within hours. Mycobacteria Most require 2 12 weeks to grow in special conditions. Some can only be cultured using animal inoculation. Fungi Require nutrients and humidity. ]Most require 7 20 days to grow, except yeast grows very quickly on routine media. Viruses and chlamydia Require special cell cultures days for growth depending the virus.

13 Examples of Culture Plates Dept. Micro., Mt. Sinai Hosp. BioMérieux, Inc.

14 Determination of the MIC Harvey & Champe. Microbiology Lippincott s Illustrated Reviews. 2001

15 ges/disk-diffusion.jpg

16 CLSI Interpretive Criteria Susceptible: S category infection due to the strain may be treated with dosage of agent recommended for that infection. Intermediate: I category isolates with MICs usually attainable in blood/tissue but response may be lower. Clinical applicability where drugs are concentrated or when high dose can be given. Also includes buffer zone for technical discrepancies. Resistant: R category strains not inhibited by usually achievable systemic concentrations using normal dosages and where clinical efficacy has not been reliable.

17 Interpretation of Susceptibility Results Larger zones do not always equal greater susceptibility Baron & Hindler. Substratum: The Microbiology Laboratory. 1998

18 Molecular Detection Detection of nucleic acids of pathogens has revolutionized the microbiology laboratory. Advantages Very high sensitivity, high specificity, reduced turn around time and irrelevance to viability Disadvantages Cost, risk of contamination, problems with validation/applicability/relevance, space requirement, expertise Current uses by DSM Pertussis Influenza (some sites) Tuberculosis 16S rrna sequencing (PFGE)

19 IMPORTANCE OF SPECIMEN COLLECTION

20 Basics of Specimen Collection Nothing is more important to the effectiveness of a laboratory than a specimen that has been appropriately selected, collected and transported Specimen collectors must: Select the correct anatomic site from which to obtain the specimen Collect the specimen using the proper technique and supplies Package the specimen in a container designed to promote survival of the causative organism and to eliminate leakage, which might pose a safety hazard; and Transport the specimen to the laboratory expeditiously or make sure that if it is stored, storage is brief, properly done, and at a temperature that will not damage the suspected organism. M. Miller, A Guide to Specimen Management in Clinical Microbiology, 1999

21 Selecting the Appropriate Site Inappropriately selected samples can lead to an erroneous diagnosis & inappropriate therapy Commensal/normal flora is background noise in many samples Levels differ by body site Lab must differentiate normal flora from pathogens Appropriately collected samples minimize normal flora Eg. Midstream urine collection, BAL vs. sputum M. Miller, A Guide to Specimen Management in Clinical Microbiology, 1999

22 The Microbiology Requisition is Critical The following information must be provided on the requisition Patient Name PHIN #/Chart # Date of Birth (Day/Month/Year) Patient Location Ordering Physician/Practitioner Collection Date

23 Specimen Labeling The following information is required directly on the specimen container: Patient name (Last name, First name) PHIN # or equivalent (unique identifier) MUST match # on the patient requisition Source of the specimen Date and Time of collection Patient Name AND PHIN # or equivalent BOTH required on specimen

24 Common Reasons Specimens are Not Accepted Specimen is leaking Specimen is not suitable for the test that was ordered Specimen was not labeled appropriately Duplicate specimen was received on the same patient during the same day Enterics (Stool culture) 3 day rule, other reasons

25 Why Not Accept All Specimens? Potential harm to the patient Treatment decisions may be based on misleading information (e.g. contaminant, specimen not belonging to the patient) Lack of benefit for the patient E.g. inappropriate specimen for the disease of interest Potential harm to the laboratory staff E.g. leaking specimens Financial reasons very low yield with certain tests E.g. duplicate specimens, stool for routine bacterial culture on patients in hospital >3 days

26 Specimen Collection Blood Culture Example Skin preparation using chlorhexidine /alcohol swab sticks these have been found to be extremely effective in minimizing the risk of contamination with skin microorganisms. Failure to disinfect the site may lead to false positive results due to contamination Underfilling/overfilling Culturing < 10 ml per bottle reduces the ability to detect a pathogen when bacteremia is present Overfilling can reduce the ability to detect a pathogen as natural inhibitors in the blood may not be appropriately diluted out Number of sites Failure to draw from 2 sites impacts ability to differentiate a true positive from contamination

27 True (+) vs. Contamination Aerobic and anaerobic bottle 1 site Aerobic & anaerobic bottle site 1 Growth of Coagulase Negative Staphylococcus No Growth? True Pathogen or Contamination Aerobic bottle site 2 Growth of Coagulase Negative Staphylococcus Represents Probable Contamination

28 Stool for Clostridium difficile Example Rejection criteria: Formed stool (unless patient has the diagnosis of toxic megacolon) Inappropriate specimen for test Samples submitted on patients < 1 year of age Potentially misleading results Prolonged transit > 72 hours Laboratory results negatively affected Samples submitted within 7 days of a previous positive test result Laboratory utilization issues Maximum of 2 stools tested in a 7 day period Laboratory utilization issues

29 INTERPRETATION OF LABORATORY RESULTS

30 Respiratory Specimens Lab screens specimen with gram stain to determine suitability and give rapid information. Lab will culture for routine bacterial pathogens Eg. S. pneumoniae, H. influenzae, M. catarrhalis, Enterobacteriaeceae, Pseudomonas, Gram-negative rods, S. aureus, β-hemolytic streptococci, enterococci, etc. Specific request and specimens needed for TB, AFB, Legionella, anaerobes, fungi, mycoplasma.

31 Eg. Viridans Group Streptococcus, Non- pathogenic Neisseria spp., Corynebacterium spp, etc.

32 Blood Cultures BC incubated for 5 days Positive cultures Gram stain result called Rapid tests as appropriate Eg. Thermostable DNase / Tube Coagulase Eg. MRSA detection All organisms reported Potential contaminants Bacillus, Corynebacterium, Propionibacterium, CNST, viridans group strep, Aerococcus spp., Micrococcus spp.

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34 Sterile fluids / Tissues CSF, aseptically obtained tissues, biopsies, fluids, bone, aspirates, prosthetic devices Gram stain result reported ASAP ~ All organisms that grow on routine media are reported If an unusual organism is expected extra testing and/or precautions may be required and laboratory must be notified in advance e.g. BSL 3 organism, slow growing organism, AFB, fungus, (anaerobes) Extent of identification may depend on likelihood of contamination

35 Urine Typical uropathogens will be identified Enterobacteriaceae, P. aeruginosa, GBS, S. aureus, S. saprophyticus, enterococci Workup of atypical organisms requires lab consult Mixed/contaminated urine, non-pathogens (skin/vaginal flora) not identified New chromogenic media reduces TAT All urines will have a quantitative culture done Report will give pathogen and quantity per litre Eg. >1x10E8/L of E. coli

36 Example of Urine Culture Report with AST results Urine Susceptibility Report Pathogen: E. coli >10 8 CFU/mL Isolate 1 MIC Breakpoint Interpretation Ampicillin >16 >16 R Pip/Tazo <16 >64 S Amox/Clav <8 >16 S Cefazolin <4 >16 S Cefuroxime <4 >16 S Gentamicin <2 >8 S TMP/SMX <2 >4 S Nitrofurantoin <32 >64 S Ciprofloxacin <1 >2 S Levofloxacin <1 >4 S

37 Stool Culture Pathogens sought are: E. coli O157:H7, Campylobacter, Shigella, Salmonella, Yersinia enterocolitica Cultures for Vibrio, Aeromonas, other E. coli, Bacillus, Clostridium, S. aureus etc... are on request only Reports: 1. Organism identified or 2. No Salmonella, Shigella, Campylobacter, Yersinia or E.coli O157 isolated C. difficile testing is by antigen detection & molecular testing Reports 1. Toxigenic C. difficile detected. This test was performed using a Health Canada cleared nucleic acid amplification assay for the detection of a segment of Clostridium difficile DNA known to be present in all known toxigenic strains of C. difficile, including A-B+ toxinotypes. 2. Test result by NAAT was invalid; please submit another stool specimen if clinically warranted. 3. No toxigenic C. difficile detected.

38 Surveillance Specimens MRSA Chromogenic media Report A methicillin resistant S. aureus (MRSA) has been isolated. Follow the MRSA Infection Control guidelines. VRE Chromogenic media, following enrichment broth Report A vancomycin resistant Enterococcus (VRE) has been isolated. Follow the VRE Infection Control guidelines. IP&C, CDC notified of all isolations

39 COLLABORATION BETWEEN INFECTION PREVENTION AND CONTROL AND THE LABORATORY

40 Improves Patient Care You know the patients You know your site s local epidemiology We know the lab protocols & what other tests we can offer

41 Increases Utility of Laboratory Results A clear understanding of what testing was performed and what the results mean improves the usefulness of the results

42 Improves Laboratory Utilization Laboratory services are expensive Appropriate test orders improve costefficiency Communication improves the laboratory s ability to plan (re: media orders, bench workflow)

43 Questions? THANK YOU