Molecular Identification and Strain Typing of MRSA

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Molecular Identification and Strain Typing of MRSA

• John W. Longshore, Ph.D., FACMG
• Director, Molecular Pathology
• Carolinas Pathology Group
• Carolinas Medical Center

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Topics to be Covered

• Laboratory detection of MRSA
• Traditional culture
• Molecular detection
• mecA
• Strain typing
• PFGE
• REP-PCR
• Summary

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Culture Detection of MRSA

• Clinical and Laboratory Standards Institute
  (CLSI) recommends
• Cefoxitin disk screen test
• Latex agglutination test for PBP2a
• 6 ug/ml oxacillin in Mueller-Hinton agar with 4
  NaCl

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Advantages of MRSA PCR

• Rapid Identification of carriers
• 2 hours PCR
• 24-72 hours Culture
• Swift implementation of appropriate interventions
• Limits unnecessary preemptive isolation
• Limits unnecessary antibiotic use
• Identifies MRSA in mixed populations
• Methicillin resistant coagulase negative Staph
• mecA negative Staph

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MRSA PCR

• Two FDA cleared products
• BD GeneOhm (Infectio Diagnostic)
• Cepheid GeneXpert
• Runs on Cepheid SmartCycler
• Real-time PCR
• Samples type is direct nasal swab
• Detects gene sequence at the junction of the
  SCCmec and orfX gene
• Kit based product that can be used with minimal
  specialized training

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MRSA PCR Procedure

• Specimen preparation
• Concentration
• Lysis-DNA extraction
• Reconstitution of molecular reagents
• Real-time PCR
• Automated results
• Full run time lt 2 hours

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Smart Cycler Instrument
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Quantification and Dynamic Range
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Real-time Analysis
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MRSA PCR vs. Culture
PPV 94 NPV 98 Detection limit 325 MRSA per
swab
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MRSA Strain Typing

• MRSA is a clonal organism
• Very useful for epidemiology
• Strain types between patients
• Monitor trends in types
• Track seasonal outbreaks
• Database of samples for long term studies
• Trace infection source
• Patient-Patient / Patient-Caregiver
• Determine if HA or CA MRSA

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USA Strain Types

• Large study by CDC in 2003
• Sma I digested bacterial DNA
• 1000 MRSA isolates
• Determine CA vs. HA strains
• Eight major strain types in US
• CA USA 300 and 400
• Resistant to beta-lactams and erythromycin
• USA 100 most common type (44)
• Multiresistant
• USA 700 both HA and CA

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Resolution of Typing Methods
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Strain Typing Methods
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Pulsed Field Gel Electrophoresis
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PFGE Results
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Repetitive Sequence PCR
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REP PCR Protocol
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Bacterial Barcodes MRSA Typing

• Based on REP PCR technique
• Minimal tech hands on time
• Relies upon either traditional culture or PCR for
  MRSA status prior to analysis
• Cant distinguish MSSA from MRSA
• MRSA library based upon CDC USA PFGE types
• Highly reproducible

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DiversiLab MRSA Library

• USA 100
• USA 200
• USA 300
• USA 400
• USA 500
• USA 600
• USA 700
• USA 800
• USA 900
• USA 1000
• USA 1100
• USA 1200
• Brazil
• Iberian

• 2
• 3
• 3
• 3
• 2
• 2
• 2
• 1
• 1
• 2
• 2
• 1
• 2
• 2

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DNA LabChip Microfluidic Electrophoresis
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Agilent 2100 System
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Strain Typing Workflow
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DiversiLab Results
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Top Match
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Careful Interpretation!
USA 100
USA 800
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Cluster Analysis
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Why Utilize Molecular Testing?

• Rapid diagnosis
• Cost effective
• Highly specific
• Improved patient care
• Provides ultimate resolution
• No outside interference from medication or
  treatments can alter the testing results