Role of the Microbiology Laboratory in Hospital Epidemiology Karen C. Carroll, M.D. Associate Professor, Pathology Adjunct Associate Professor, Infectious Diseases University of Utah Medical Center
Nosocomial Infections • Infections not incubating on admission to a health care institution (acquired during a stay) • Infections may involve patients, visitors or hospital personnel
Nosocomial Infections:Impact • Major public health problem worldwide • Affects 5-10% of patients admitted to U.S. hospitals-- 2 million patients annually • Significant annual expenditures-- $4.5 billion • Contribute significantly to morbidity and mortality • nosocomial BSI--25% attributable mortality • 20,000 deaths annually from HAP • catheter-related UTIs also contribute to risk of dying
Major Types of Nosocomial Infections Richards, MJ. 1999. Crit Care Med 27; 887.
Mid-1980’s Enterobacteriaceae S. aureus P. aeruginosa CoNS Mid-1990’s Decline in Enterobacteriaceae Increase in gram-positive cocci Emergence of fungi Recognition of viruses Nosocomial Infections:Changing Microbiology
Selected Antimicrobial Resistant Pathogens: Nosocomial Infections in ICU Patients
Role of the Microbiology Laboratory • Grow and detect microbial pathogens • Identify causative organisms rapidly and accurately to species level • Perform accurate susceptibility testing • Recognize limitations of automated methods • Provide supplemental testing for problem bacteria • Recognize and survey for MDR organisms
Glycopeptide Intermediate S. aureus All labs should have a procedure for selection of S. aureus strains for additional testing • S. aureus with MICs > 4 g/ml • S. aureus with MICs > 8g/ml • Select and test all MRSA • Select isolates growing on screening agar • BHI agar containing 6 g/ml vancomycin • Use inoculum of 106 cfu/ml
Glycopeptide Intermediate S. aureus:Testing • Disk diffusion is unacceptable • MIC testing method should be used • broth microdilution • agar dilution • agar gradient diffusion • Incubate for full 24h at 35° C • Use S. aureus ATCC 29213 as neg. control strain • Send to State Lab/CDC for confirmation • SEARCH@cdc.gov
Role of the Microbiology Lab Timely reporting • “Early warning system” • Infection control critical values • Positive AFB smears and Mtb cultures • VRE • MRSA. GISA • Legionella • Salmonella/shigella • Multi-drug resistant GNR • Data summaries, monitoring trends
Direct Infection-Control Related Functions: Micro Lab • Participate as a member of the infection control committee • Ensures communication • Enhances education • Allows for allocation of resources • Organize and report microbiology data • Antibiograms • Customized epidemiological reports • Store data and isolates
Direct Infection-Control Related Functions:Micro Lab • Collaborate with IC personnel on outbreak investigations • Perform standard typing tests • Serve as an educational resource • basic microbiology training • periodic updates: changes in technology, taxonomy
Application of Typing Techniques • Epidemiological investigations • increase in prevalence of infections due to a particular species • clusters of patients • identification of isolates that have a distinctive susceptibility pattern • Distinguishing relapse from re-infection • Establishing clonality of isolates
Epidemiologic Typing Systems Criteria for evaluation • Typeability • Reproducibility • Discriminatory power • Ease of interpretation • Ease of performance
Epidemiologic Typing Systems:Classification Phenotypic • Traditional • biotyping • antimicrobial susceptibility testing • serotyping • bacteriophage typing • bacteriocin typing • Protein-based • multi-locus enzyme electrophoresis • polyacrylamide gel electrophoresis of cellular proteins • Immunoblot fingerprinting
Phenotypic Typing Methods Limitations • Influenced by environmental selective pressure • unstable antigenic traits • alterations in expression of traits being assessed • Labor-intensive • Impractical • Slow • Lack discriminatory power
Epidemiologic Typing SystemsClassification Genotypic methods • Plasmid analysis • Restriction endonuclease analysis chromosomal DNA (REA) • Southern blot analysis of RFLP • Ribotyping • PFGE • PCR techniques • Sequence analysis • Gene expression--microarrays
Genotypic Typing Methods Limitations • Patterns generated may be complex and difficult to interpret • Technically demanding • Methodology/ interpretation is not standardized
PFGE Principles • Variation of conventional agarose gel electrophoresis • Suspension of organism is embedded in agarose plugs to minimize shearing of DNA • DNA is cut with restriction enzymes that have infrequent recognition sites • Larger pieces of DNA are separated by shifting direction of current frequently • 5-20 fragments ranging in size from 10kb to 800 kb in length are generated
Tenover Criteria for PFGE Interpretation • Identical isolates--all bands match • Isolates are subtypes--patterns differ by 1 to 3 bands • Isolates are possibly related--patterns differ by 4-6 bands • Isolates are unrelated--patterns differ by more than 6 bands Tenover FC, et. al. J Clin Microbiol 33:2233, 1995.
Modified Tenover Criteria • < 3 differences in restriction-fragment positions • could have occurred by a single genetic event • may represent subtypes of the same strain • >3 restriction differences in restriction fragment positions • less likely to be epidemiologically related Goering RV. In Rapid Detection of Infectious Agents, Specter, et.al. (eds), Plenum Press, New York, 1998, p.131.
University of Utah Medical CenterVRE Outbreak • First isolate--mediastinal surgical wound • 10 subsequent cases over 15 months in MICU • Source of isolates • blood--4 • urine or stool--4 • other sites--2
University of Utah Medical CenterVRE Outbreak Characteristics of Isolates • All isolates were E. faecium, Van B phenotype • Resistant to ampicillin, vancomycin, ofloxacin, imipenem • HLR streptomycin, not gentamicin • Susceptible to teicoplanin,dalfopristin-quinupristin, chloramphenicol
PFGE: Utility • Community Outbreaks • Hospital clusters • Laboratory contamination • Resolution of pseudo-outbreaks • Individual patient management • Relapse vs. Re-infection • Demonstration of persistence of infection • Distinction between contamination vs.. infection
Advantages Patterns easier to interpret compared to other techniques Highly reproducible Excellent discriminatory power Theoretically all bacteria are typeable, some fungi as well Disadvantages Cost of equipment Tedious Slow Certain organisms may not be typeable e.g.. C. difficile, Aspergillus sp PFGE
RiboPrinter System Features • Eight samples at a time; 32 samples/shift • Results in eight hours • Completely automated • Results stored electronically • Electronic sharing of data
PFGE vs. Ribotyping for VRE Characterization Study Objectives • Compare PFGE vs. ribotyping using two restriction enzymes for VRE • 94 total VRE isolates • Late 1995 - mid-2000 • outbreak and non-outbreak isolates from University of Utah
Automated Ribotyping:VRE Study • Two restriction endonucleases simultaneously (1:1 mix of AseI and BamHI) • DNA electrophoresed and transferred to nylon membrane (Southern Blot) • Probed for rRNA operon-specific DNA fragments • Band pattern captured by CCD camera, normalized and stored in computer memory • TAT of < 24 hours
Results • 24 unique PFGE types • 26 unique ribotype patterns • Multiple PFGE types - same ribotype • 2 instances • Single PFGE type - multiple ribotypes • 3 instances
15.00 40.00 1.00 2.00 3.00 4.00 6.00 8.00 00-116-10237 . . . . 16 17 00-111-08430 . . . . 11 15 Outbreak vs. Non-outbreak
Study Conclusions • Ribotyping using double RE digest comparable to PFGE • PFGE able to discriminate subtypes • Ribotyping uses higher degree of automation • Ribotyping has faster TAT • more amenable to real-time characterization • Overall costs / sample are comparable
rep-PCRCore Technology BACTERIA 2 BACTERIA 1 Primer Primer Primer DNA Primer DNA DNA DNA Primer DNA DNA Rep-PCR DNA Primer Primer
Strain Differentiation With reproducible fingerprints
Epidemiologic Typing MethodsBasic Principles • Perform only with clear objectives • Variability exists in all methods • evaluate all implicated isolates simultaneously • compare to epidemiologically unrelated control isolates • Demonstrate not only relatedness of clustered isolates, but differences from isolates not involved epidemiologically
Integrated Infection Control Program • Active surveillance of high-risk patients • Incorporation of molecular typing into routine surveillance • Weekly meetings to discuss trends • Information used to implement appropriate infection control practice • education of staff • physical barriers Peterson LR and Noskin GA. Emerg Infect Dis 7:306, 2001.
Integrated Infection Control Program:Impact • Nosocomial infections decreased from 6.49/1,000 pt. days to 5.60/1,000 pt. days • Percentage of patients with nosocomial infections dropped by 23% • Costs avoided averaged more than $2 million/yr. • Costs for the program paid by the hospital • initial equipment/remodeling $180,050 • $400,000 annual costs for med techs Peterson LR and Noskin GA. Emerg Infect Dis 7:306, 2001.
Infection Control Shift toward focused surveillance ICUs devices antimicrobial resistance Control strategies are more proactive active intervention control of resistance Microbiology Labs Increasingly complex and demanding work increasing resistance emerging pathogens new technology Monitoring resistance Implementation of molecular epidemiology Expanded Roles of Infection Control and Microbiology Labs