Role of the Role of the Microbiology Microbiology

Laboratory in Hospital Laboratory in Hospital EpidemiologyEpidemiology

Karen C. Carroll, M.D.Karen C. Carroll, M.D.

Associate Professor, PathologyAssociate Professor, Pathology

Adjunct Associate Professor, Adjunct Associate Professor, Infectious DiseasesInfectious Diseases

University of Utah Medical CenterUniversity of Utah Medical Center

Nosocomial InfectionsNosocomial Infections

Infections not incubating on Infections not incubating on admission to a health care admission to a health care institution (acquired during a stay)institution (acquired during a stay)

Infections may involve patients, Infections may involve patients, visitors or hospital personnelvisitors or hospital personnel

Nosocomial Nosocomial Infections:ImpactInfections:Impact

Major public health problem worldwideMajor public health problem worldwide Affects 5-10% of patients admitted to U.S. Affects 5-10% of patients admitted to U.S.

hospitals-- 2 million patients annuallyhospitals-- 2 million patients annually Significant annual expenditures-- $4.5 Significant annual expenditures-- $4.5

billion billion Contribute significantly to morbidity and Contribute significantly to morbidity and

mortalitymortality– nosocomial BSI--25% attributable mortalitynosocomial BSI--25% attributable mortality– 20,000 deaths annually from HAP20,000 deaths annually from HAP– catheter-related UTIs also contribute to risk of catheter-related UTIs also contribute to risk of

dyingdying

Major Types of Nosocomial Major Types of Nosocomial InfectionsInfections

0

5

10

15

20

25

30

35

Overall ICU

UTIPneumoniaSWIBloodstreamOther

Richards, MJ. 1999. Crit Care Med 27; 887.

Nosocomial Infections:Nosocomial Infections:Changing MicrobiologyChanging Microbiology

Mid-1980’sMid-1980’s– EnterobacteriaceEnterobacteriace

aeae– S. aureusS. aureus– P. aeruginosaP. aeruginosa– CoNSCoNS

Mid-1990’sMid-1990’s– Decline in Decline in

EnterobacteriaceEnterobacteriaceaeae

– Increase in gram-Increase in gram-positive coccipositive cocci

– Emergence of Emergence of fungifungi

– Recognition of Recognition of virusesviruses

Major Pathogens Associated with Major Pathogens Associated with each Category of Nosocomial each Category of Nosocomial

InfectionsInfections

BSIPathogen (%)

PneumoniaPathogen (%)

SSIPathogen (%)

UTIPathogen (%)

CoNS 39 S. aureus 17 Enterococci 17 E. coli 19

S. aureus 12 P. aerug 16 CoNS 12 C. albicans 15

Enterococci 11 Enterobact 11 P. aerug 10 Enterococci 14

Candida sp. 11 K. pneumon 7 S. aureus 9 P. aerug. 10

Resistance Pattern 2000 ResistanceRate (%)

1995-1999Resistance Rates*

(%)

% Increase inResistance

Vanco/Enterococci 26 14-25 31

Meth/S. aureus 55 33-51 29

Meth/CoNS 87 85-88 1

3rd ceph/E. coli 3 2-5 15

3rd ceph/K. pneumo 11 9-11 5

Imipenem/P. aerug 18 12-16 23

Quinolone/P. aerug 27 12-23 53

3rd ceph/P. aerug 26 19-23 243rd ceph/Enterobact 35 33-37 -1

*Mean rates +/- one standard deviation. CDC website NNIS ICU data. AJIC 29:410, 2001.

Selected Antimicrobial Resistant Pathogens: Nosocomial Infections in ICU Patients

Role of the Microbiology Role of the Microbiology LaboratoryLaboratory

Grow and detect microbial pathogensGrow and detect microbial pathogens Identify causative organisms rapidly Identify causative organisms rapidly

and accurately to species leveland accurately to species level Perform accurate susceptibility testingPerform accurate susceptibility testing

– Recognize limitations of automated Recognize limitations of automated methodsmethods

– Provide supplemental testing for problem Provide supplemental testing for problem bacteriabacteria

– Recognize and survey for MDR organismsRecognize and survey for MDR organisms

Glycopeptide Glycopeptide Intermediate Intermediate

S. aureusS. aureus

All labs should have a procedure for All labs should have a procedure for selection of selection of S. aureusS. aureus strains for strains for additional testingadditional testing– S. aureusS. aureus with MICs with MICs >> 4 4 g/mlg/ml– S. aureusS. aureus with MICs with MICs >> 8 8g/mlg/ml– Select and test all MRSASelect and test all MRSA– Select isolates growing on screening agarSelect isolates growing on screening agar

BHI agar containing 6 BHI agar containing 6 g/ml vancomycing/ml vancomycin Use inoculum of 10Use inoculum of 1066 cfu/ml cfu/ml

Glycopeptide Glycopeptide Intermediate Intermediate

S. aureus:S. aureus:TestingTesting

Disk diffusion is unacceptableDisk diffusion is unacceptable MIC testing method should be usedMIC testing method should be used

– broth microdilutionbroth microdilution– agar dilutionagar dilution– agar gradient diffusionagar gradient diffusion

Incubate for full 24h at 35° CIncubate for full 24h at 35° C Use Use S. aureusS. aureus ATCC 29213 as neg. ATCC 29213 as neg.

control straincontrol strain Send to State Lab/CDC for Send to State Lab/CDC for

confirmationconfirmation– SEARCH@cdc.govSEARCH@cdc.gov

Role of the Role of the Microbiology LabMicrobiology Lab

Timely reportingTimely reporting ““Early warning system”Early warning system” Infection control critical valuesInfection control critical values

– Positive AFB smears and Mtb culturesPositive AFB smears and Mtb cultures– VREVRE– MRSA. GISAMRSA. GISA– Legionella Legionella – Salmonella/shigellaSalmonella/shigella– Multi-drug resistant GNRMulti-drug resistant GNR

Data summaries, monitoring Data summaries, monitoring trendstrends

Direct Infection-Control Direct Infection-Control Related Functions: Micro LabRelated Functions: Micro Lab

Participate as a member of the Participate as a member of the infection control committeeinfection control committee– Ensures communicationEnsures communication– Enhances educationEnhances education– Allows for allocation of resourcesAllows for allocation of resources

Organize and report microbiology dataOrganize and report microbiology data– AntibiogramsAntibiograms– Customized epidemiological reportsCustomized epidemiological reports

Store data and isolatesStore data and isolates

Direct Infection-Control Direct Infection-Control Related Functions:Micro LabRelated Functions:Micro Lab

Collaborate with IC personnel on Collaborate with IC personnel on outbreak investigationsoutbreak investigations

Perform standard typing testsPerform standard typing tests Serve as an educational resourceServe as an educational resource

– basic microbiology trainingbasic microbiology training– periodic updates: changes in periodic updates: changes in

technology, taxonomytechnology, taxonomy

Role of the Microbiology Lab Role of the Microbiology Lab in Investigation of an in Investigation of an

OutbreakOutbreak

Investigative Step Laboratory’s RoleRecognize the problem Form case definition Look for additional cases Calculate rates

Laboratory surveillance Communication Microbiologic confirmation Store data and isolates

Characterize the outbreak Who Where When What

Characterize isolates Type isolates Assess number and location

Role of the Microbiology Lab Role of the Microbiology Lab in Investigation of an in Investigation of an

OutbreakOutbreak

Investigative Step Laboratory’s RoleConsider possible causes

Define mode of transmission Identify potential reservoirs Identify potential vectors

Conduct supplementary studies Cultures from personnel, patients’ environment

Control/terminate the outbreak Define/implement control measures Evaluate efficacy of control measures Continued surveillance

Adjust lab procedures tosupport control activities Continue lab surveillance Store isolates Maintain communication

Application of Typing Application of Typing TechniquesTechniques

Epidemiological investigations Epidemiological investigations – increase in prevalence of infections due increase in prevalence of infections due

to a particular speciesto a particular species– clusters of patients clusters of patients – identification of isolates that have a identification of isolates that have a

distinctive susceptibility patterndistinctive susceptibility pattern Distinguishing relapse from re-Distinguishing relapse from re-

infectioninfection Establishing clonality of isolatesEstablishing clonality of isolates

Epidemiologic Typing Epidemiologic Typing SystemsSystems

Criteria for evaluationCriteria for evaluation TypeabilityTypeability ReproducibilityReproducibility Discriminatory powerDiscriminatory power Ease of interpretationEase of interpretation Ease of performanceEase of performance

Epidemiologic Typing Epidemiologic Typing Systems:Systems:

ClassificationClassificationPhenotypicPhenotypic TraditionalTraditional

– biotypingbiotyping– antimicrobial susceptibility testingantimicrobial susceptibility testing– serotypingserotyping– bacteriophage typingbacteriophage typing– bacteriocin typingbacteriocin typing

Protein-basedProtein-based– multi-locus enzyme electrophoresismulti-locus enzyme electrophoresis– polyacrylamide gel electrophoresis of cellular polyacrylamide gel electrophoresis of cellular

proteinsproteins– Immunoblot fingerprintingImmunoblot fingerprinting

Phenotypic Typing Phenotypic Typing MethodsMethods

LimitationsLimitations Influenced by environmental selective Influenced by environmental selective

pressurepressure– unstable antigenic traitsunstable antigenic traits– alterations in expression of traits being alterations in expression of traits being

assessedassessed Labor-intensiveLabor-intensive ImpracticalImpractical SlowSlow Lack discriminatory powerLack discriminatory power

Epidemiologic Typing Epidemiologic Typing SystemsSystems

ClassificationClassificationGenotypic methodsGenotypic methods Plasmid analysisPlasmid analysis Restriction endonuclease analysis Restriction endonuclease analysis

chromosomal DNA (REA)chromosomal DNA (REA) Southern blot analysis of RFLPSouthern blot analysis of RFLP RibotypingRibotyping PFGEPFGE PCR techniquesPCR techniques Sequence analysisSequence analysis Gene expression--microarraysGene expression--microarrays

Genotypic Typing Genotypic Typing MethodsMethods

LimitationsLimitations Patterns generated may be Patterns generated may be

complex and difficult to interpretcomplex and difficult to interpret Technically demandingTechnically demanding Methodology/ interpretation is not Methodology/ interpretation is not

standardizedstandardized

PFGE PrinciplesPFGE Principles Variation of conventional agarose gel Variation of conventional agarose gel

electrophoresiselectrophoresis Suspension of organism is embedded in Suspension of organism is embedded in

agarose plugs to minimize shearing of agarose plugs to minimize shearing of DNADNA

DNA is cut with restriction enzymes that DNA is cut with restriction enzymes that have infrequent recognition sites have infrequent recognition sites

Larger pieces of DNA are separated by Larger pieces of DNA are separated by shifting direction of current frequentlyshifting direction of current frequently

5-20 fragments ranging in size from 10kb 5-20 fragments ranging in size from 10kb to 800 kb in length are generatedto 800 kb in length are generated

Pulsed-Field Gel Electrophoresis (PFGE)

Tenover Criteria for PFGE Tenover Criteria for PFGE InterpretationInterpretation

Identical isolates--all bands matchIdentical isolates--all bands match Isolates are subtypes--patterns Isolates are subtypes--patterns

differ by 1 to 3 bandsdiffer by 1 to 3 bands Isolates are possibly related--Isolates are possibly related--

patterns differ by 4-6 bandspatterns differ by 4-6 bands Isolates are unrelated--patterns Isolates are unrelated--patterns

differ by more than 6 bands differ by more than 6 bands Tenover FC, et. al. J Clin Microbiol 33:2233, 1995.Tenover FC, et. al. J Clin Microbiol 33:2233, 1995.

Modified Tenover Modified Tenover CriteriaCriteria

<< 3 differences in restriction-fragment 3 differences in restriction-fragment positionspositions– could have occurred by a single genetic could have occurred by a single genetic

eventevent– may represent subtypes of the same may represent subtypes of the same

strainstrain >3 restriction differences in restriction >3 restriction differences in restriction

fragment positionsfragment positions– less likely to be epidemiologically relatedless likely to be epidemiologically related

Goering RV. In Goering RV. In Rapid Detection of Infectious AgentsRapid Detection of Infectious Agents, Specter, , Specter, et.al. (eds), Plenum Press, New York, 1998, p.131.et.al. (eds), Plenum Press, New York, 1998, p.131.

University of Utah Medical University of Utah Medical CenterCenter

VRE OutbreakVRE Outbreak

First isolate--mediastinal surgical First isolate--mediastinal surgical wound wound

10 subsequent cases over 15 10 subsequent cases over 15 months in MICUmonths in MICU

Source of isolatesSource of isolates– blood--4blood--4– urine or stool--4urine or stool--4– other sites--2other sites--2

University of Utah Medical University of Utah Medical CenterCenter

VRE OutbreakVRE Outbreak

Characteristics of IsolatesCharacteristics of Isolates All isolates were All isolates were E. faecium,E. faecium, Van B Van B

phenotypephenotype Resistant to ampicillin, vancomycin, Resistant to ampicillin, vancomycin,

ofloxacin, imipenemofloxacin, imipenem HLR streptomycin, not gentamicinHLR streptomycin, not gentamicin Susceptible to teicoplanin,dalfopristin-Susceptible to teicoplanin,dalfopristin-

quinupristin, chloramphenicolquinupristin, chloramphenicol

PFGE: UtilityPFGE: Utility Community OutbreaksCommunity Outbreaks Hospital clustersHospital clusters Laboratory contaminationLaboratory contamination Resolution of pseudo-outbreaksResolution of pseudo-outbreaks Individual patient managementIndividual patient management

– Relapse vs. Re-infectionRelapse vs. Re-infection– Demonstration of persistence of Demonstration of persistence of

infectioninfection– Distinction between contamination Distinction between contamination

vs.. infectionvs.. infection

PFGEPFGE

AdvantagesAdvantages Patterns easier to Patterns easier to

interpret compared to interpret compared to other techniquesother techniques

Highly reproducibleHighly reproducible Excellent Excellent

discriminatory powerdiscriminatory power Theoretically all Theoretically all

bacteria are typeable, bacteria are typeable, some fungi as wellsome fungi as well

DisadvantagesDisadvantages Cost of equipmentCost of equipment TediousTedious SlowSlow Certain organisms Certain organisms

may not be typeable may not be typeable e.g..e.g..

CC. . difficiledifficile, , Aspergillus spAspergillus sp

RiboPrinterRiboPrinter®® Microbial Microbial Characterization SystemCharacterization System

RiboPrinter System RiboPrinter System FeaturesFeatures

Eight samples at a time; 32 Eight samples at a time; 32 samples/shiftsamples/shift

Results in eight hoursResults in eight hours Completely automatedCompletely automated Results stored electronicallyResults stored electronically Electronic sharing of dataElectronic sharing of data

PFGE vs. Ribotyping for PFGE vs. Ribotyping for VRE CharacterizationVRE Characterization

Study ObjectivesStudy Objectives Compare PFGE vs. ribotyping using Compare PFGE vs. ribotyping using

two restriction enzymes for VREtwo restriction enzymes for VRE

94 total VRE isolates94 total VRE isolates– Late 1995 - mid-2000Late 1995 - mid-2000– outbreak and non-outbreak isolates outbreak and non-outbreak isolates

from University of Utahfrom University of Utah

Automated Ribotyping:VRE Automated Ribotyping:VRE StudyStudy

Two restriction endonucleases Two restriction endonucleases simultaneously (1:1 mix of simultaneously (1:1 mix of AseAseI and I and BamHBamHI)I)

DNA electrophoresed and transferred to DNA electrophoresed and transferred to nylon membrane (Southern Blot)nylon membrane (Southern Blot)

Probed for rRNA operon-specific DNA Probed for rRNA operon-specific DNA fragmentsfragments

Band pattern captured by CCD camera, Band pattern captured by CCD camera, normalized and stored in computer normalized and stored in computer memorymemory

TAT of < 24 hoursTAT of < 24 hours

Riboprint PatternsRiboprint Patterns

ResultsResults

24 unique PFGE types24 unique PFGE types 26 unique ribotype patterns26 unique ribotype patterns

Multiple PFGE types - same Multiple PFGE types - same ribotyperibotype– 2 instances2 instances

Single PFGE type - multiple Single PFGE type - multiple ribotypesribotypes– 3 instances3 instances

Outbreak vs. Non-Outbreak vs. Non-outbreakoutbreak

1.00

2.00

3.00

4.00

6.00

8.00

15.0

0

40.0

0

00-116-10237

00-111-08430

.

.

.

.

.

.

.

.

16

11

17

15

§ Non-outbreak associated strain † Outbreak associated strain

Isolate # PFGE RiboGroup Type

†

§

Able to differentiate non-outbreak strain during an outbreak period. During the outbreakinvolving PFGE type 11, PFGE type 16 was isolated. The RiboPrinter® System, using AseI andBamHI simultaneously, was able to differentiate outbreak vs. non-outbreak associated strainsthat were temporally related.

PFGE RiboGroup

Study ConclusionsStudy Conclusions Ribotyping using double RE digest Ribotyping using double RE digest

comparable to PFGEcomparable to PFGE PFGE able to discriminate subtypesPFGE able to discriminate subtypes Ribotyping uses higher degree of Ribotyping uses higher degree of

automationautomation Ribotyping has faster TATRibotyping has faster TAT

– more amenable to real-time characterizationmore amenable to real-time characterization Overall costs / sample are comparableOverall costs / sample are comparable

rep-PCRrep-PCR Core TechnologyCore TechnologyBACTERIA 1 BACTERIA 2

Rep-PCR

Primer

Primer

DNA

DNA Primer

DNA

Primer Primer

Primer

Primer

DNA

DNA

DNA

DNA

Strain DifferentiationStrain DifferentiationWith reproducible fingerprints

Comparison of Competing Comparison of Competing TechnologiesTechnologiesCultures PFGE AFLP Ribotyping repPCR

SubspeciesDiscrimination

X X X X

Low cost X X

Universalequipment

X X

Rapid X

UniversalPrimers

X

Reproducible X X X

Databasecapable

X X X

DiversiMap Phase I: Gel DiversiMap Phase I: Gel AnalysisAnalysis

Epidemiologic Typing Epidemiologic Typing MethodsMethods

Basic PrinciplesBasic Principles

Perform only with clear objectivesPerform only with clear objectives Variability exists in all methodsVariability exists in all methods

– evaluate all implicated isolates evaluate all implicated isolates simultaneouslysimultaneously

– compare to epidemiologically compare to epidemiologically unrelated control isolatesunrelated control isolates

Demonstrate not only relatedness Demonstrate not only relatedness of clustered isolates, but of clustered isolates, but differences from isolates not differences from isolates not involved epidemiologicallyinvolved epidemiologically

Integrated Infection Integrated Infection Control ProgramControl Program

Active surveillance of high-risk patientsActive surveillance of high-risk patients Incorporation of molecular typing into Incorporation of molecular typing into

routine surveillanceroutine surveillance Weekly meetings to discuss trendsWeekly meetings to discuss trends Information used to implement Information used to implement

appropriate infection control practiceappropriate infection control practice– education of staff education of staff – physical barriersphysical barriers

Peterson LR and Noskin GA. Emerg Infect Dis 7:306, 2001.Peterson LR and Noskin GA. Emerg Infect Dis 7:306, 2001.

Integrated Infection Integrated Infection Control Control

Program:ImpactProgram:Impact Nosocomial infections decreased from Nosocomial infections decreased from

6.49/1,000 pt. days to 5.60/1,000 pt. days6.49/1,000 pt. days to 5.60/1,000 pt. days Percentage of patients with nosocomial Percentage of patients with nosocomial

infections dropped by 23%infections dropped by 23% Costs avoided averaged more than $2 Costs avoided averaged more than $2

million/yr.million/yr. Costs for the program paid by the hospitalCosts for the program paid by the hospital

– initial equipment/remodeling $180,050initial equipment/remodeling $180,050– $400,000 annual costs for med techs$400,000 annual costs for med techsPeterson LR and Noskin GA. Emerg Infect Dis 7:306, Peterson LR and Noskin GA. Emerg Infect Dis 7:306,

2001.2001.

Expanded Roles of Infection Expanded Roles of Infection Control and Microbiology Control and Microbiology

LabsLabs

Infection ControlInfection Control Shift toward Shift toward

focused focused surveillancesurveillance– ICUsICUs– devicesdevices– antimicrobial antimicrobial

resistanceresistance Control strategies Control strategies

are more proactiveare more proactive– active interventionactive intervention– control of resistancecontrol of resistance

Microbiology LabsMicrobiology Labs Increasingly complex Increasingly complex

and demanding workand demanding work– increasing resistanceincreasing resistance– emerging pathogensemerging pathogens– new technologynew technology

Monitoring resistanceMonitoring resistance Implementation of Implementation of

molecular molecular epidemiologyepidemiology