Upload
darcy-rice
View
215
Download
2
Tags:
Embed Size (px)
Citation preview
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– [email protected]@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