NOSOCOMIAL INFECTIONS NEW METHODS OF DETECTION

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NOSOCOMIAL INFECTIONSNEW METHODS OF DETECTION
CHARACTERIZATION

• Thomas Wisniewski
• Microbiology Supervisor
• VAMC, Milwaukee

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OBJECTIVES

• UPON COMPLETION ATTENEDEES WILL BE ABLE TO
• RECOGNIZE MOST COMMON ORGANISMS ASSOCIATED WITH
  NOSOCOMIAL INFECTIONS
• DISCUSS CURRENT METHODS USED TO ISOLATE AND
  IDENTIFY THESE MICROBES
• VALIDATE THE USE OF METHODS
• DISCUSS QC REQUIREMENTS OF THESE METHODS
• APPLY NEW METHODS TO STRAIN TYPE THESE MICROBES

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BUGS OF CONCERN

• METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS
  (MRSA)
• VANCOMYCIN RESISTENT ENTEROCOCCUS (VRE)
• CLOSTRIDIUM DIFFICILE
• EXTENDED SPECTRUM BETA-LACTAMASE (ESBL) ORGANISMS
• MOLDS (IN TRANSPLANT FACILITIES)
• OTHERS

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MRSA SCREENS

• CLASSIC METHOD
• ROUTINE CULTURE PLATED ON STANDARD MICROBIOLOGY
  MEDIA
• BIOCHEMICAL ID SENSITIVITY TESTING
• IMPROVED CULTURE METHODS
• BROTH ENRICHMENT
• SELECTIVE MEDIA
• USE DIFFERENT ANTIBIOTICS
• REAL TIME PCR

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Conventional Culture Method (BD SLIDE)

• CNA plate (35C, 24-48h) BHI broth w/ 6.5 NaCl
  (35C, 24h)
• read for S. aureus (catalase, coagulase)
• Screen for resistance to Penicillinase-Resistant
  Penicillins
• - Mueller Hinton agar w/ 4 NaCl 6 µg/ml
  Oxacillin (35C, 24h)
• Interpretation Positive any growth
  indicates RESISTANCE
• Negative no growth
  indicates susceptibility

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CHROMagar MRSA (BD SLIDE)
Nasal Swab
CNA (35C, 24-48h)
CHROMagar MRSA
Screen for Oxacillin R as in flowchart 1
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SELECTIVE MEDIA

• BBL CHROMAGAR MRSA

• BIORAD MRSA SELECT

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PROS AND CONS

• MRSA CHROMAGAR
• PROS
• EASE OF READING
• NON-BATCHED
• SENSITIVITY
• CONS
• 48 HOUR INCUBATION
• STORAGE
• NASAL SWABS ONLY

• MRSA SELECT
• PROS
• EASE OF READING
• 24 HOUR INCUBATION
• SENSITIVITY
• CONS
• BATCHING REQUIRED
• STORAGE
• NASAL SWABS ONLY

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VALIDATION

• CULTURE COMPARISON
• DUAL SWAB COLLECTION SYSTEM
• SETUP ON CONVENTIONAL AND SELECT MEDIA
• COMPLETE ID AND SENSITIVITY TESTING ON BOTH TYPES
  OF MEDIA

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QUALITY CONTROL

• HANDLE LIKE ANY OTHER SUSCEPTIBILITY TESTING
• 20 DAY CONSECUTIVE DAY TESTING WITH POSITIVE
  NEGATIVE CONTROLS
• PERFORM WITH EACH NEW SHIPMENT AND LOT
• PERFORM WEEKLY

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MOLECULAR METHODS

• 2 PCR METHODS FDA APPROVED
• GeneOhm BY BD DIAGNOSTICS
• GeneXpert BY CEPHEID DIAGNOSTICS
• BOTH METHODS ARE REAL TIME PCR
• BOTH METHODS UTILIZE SMART CYCLER TECHNOLOGY

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Test Procedure (BD SLIDE)
Nasal Swab
lt2 hours Total Process Time
SmartCycler Detection and Results (60 min)
Sample Buffer
Lysis Tube
SmartCycler Tube
DNA
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SUPPORT DATAVAN HAL et al JCM 2007
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VALIDATION QC

• VALIDATION
• DUAL SWAB SYSTEM REQUIRED
• SETUP ON PLATES AND DO MOLECULAR
• COMPARE DATA
• QC
• POSITIVE AND NEGATIVE CONTROL TO BE RUN WITH EACH
  ASSAY RUN

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CEPHEID GENEXPERT
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VALIDATION QC

• VALIDATION
• DUAL SWAB SYSTEM REQUIRED
• SETUP ON PLATES AND DO MOLECULAR
• COMPARE DATA
• QC
• INTERNAL CONTROLS WITH EACH KIT
• NEED TO RUN EXTERNAL CONTROLS ONLY WITH EACH
  SHIPMENT OR NEW LOT

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METHOD COMPARISONMehta et al Poster D880 ICCAC
Chicago Ill.2007
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PROS CONS

• GeneOhm
• PROS
• SENSITIVE
• HIGH VOLUME
• COST
• RAPID
• CONS
• BATCHING
• RUN TIME
• MANUAL
• REAGENT USE

• GeneXpert
• PROS
• SENSITIVE
• AUTOMATED
• SELF CONTAINED
• RAPID
• RUN TIME
• INSTRUMENT SIZE
• CONS
• COST

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VANCOMYCIN SCREENS

• CULTURE METHODOLOGY ONLY
• CLASSIC PLATING
• GROW ON PLATES
• PERFORM ID AND DO SUSCEPTIBILITY TESTING
• EITHER CONPLETE SENSITIVITY OR LOOK FOR GENES
  (VAN A, VAN B, VAN C)
• SELECTIVE MEDIA
• VANCOMYSIN SCREEN AGAR
• CAMPYLOBACTER MEDIUM

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NEW METHODS--VRE

• AMPLIFIED MOLECULAR METHODS
• HOME BREWED PCR
• LOOKING FOR VAN A AND VAN B
• SENSITIVITIES OF 68 TO 87
• INHIBITORS PRESENT IN STOOL
• IMPROVE SENSITIVITY WITH INCLUSION OF SELECTIVE
  BROTH AND THEN PCR
• FDA APPROVED METHODS
• BOTH COMMERCIAL COMPANIES IN PROCESS OF DEVELOPING

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VALIDATION AND QC

• FOR CULTURE MEDIASIMILAR TO MRSA
• COMPARISON TO STANDARD CULTURE, IDENTIFICATION
  SUSCEPTIBILITY
• VALIDATION REQUIRES CLINICAL CORELATION
• ADD STATEMENT ABOUT NON-FDA APPROVED
• ADDITIONAL CAP REQUIREMENTS

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CLOSTRIDIUM DIFFICILE

• LOOKING FOR THE TOXIN NOT JUST THE ORGANISM
• GOLD STANDARD---CYTOTOXIC EFFECT ON CELL CULTURES
• EIA TESTING MOST COMMON
• LOOKING FOR TOXIN A OR TOXIN A/B
• NEW STRAIN HAS BINARY TOXIN
• LATEX AGGLUTINATION TESTS
• 2 STEP EVAL USING BIOSITE TRIAGE
• STOOL CULTURE NOT RECOMMEND

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ESLBL PRODUCERS OTHER BUGS

• ESBL RESISTANCE
• MEDIATED BY PLASMID MEDIATED GENE
• CALLED TEM 1 SHV 1
• FOUND IN E. COLI, PROTEUS AND KLEB
• PRESENTLY NEED TO CULTURE AND THEN TEST FOR
  RESISTANCE
• OTHERS
• ASPERGILLUS
• ACINETOBACTER

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STRAIN TYPING

• OLD METHODS
• BIOCHEMICAL BIOTYPING
• SUSCEPTIBILITY BIOTYPING
• MRSA AND INDUCIBLE RESISTANCE
• NEW MOLECULAR METHODS
• PULSE FIELD GEL-ELECTROPHORESIS
• COMPARE ISOLATE GENETICS ON GEL
• EACH GEL IS UNIQUE
• MICROFLUIDIC ELECTROPHORESIS
• TECHNOLOGY ALLOWS FOR INTERRUN COMPARISON

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DIVERSILAB

• repPCR TECHNOLOGY
• rep-PCR primers bind to many specific repetitive
  sequences interspersed throughout the genome.
  Multiple fragments of various lengths are
  amplified.
• Fragments are separated using microfluidics
  electrophoresis
• As the fragments separate through the chip they
  pass over a laser thereby creating a graph of
  fluorescence intensity over time.
• This graph translates into the fingerprint
  pattern of the sample.

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STRAIN ANALYSIS

• DENDROGRAM The DiversiLab dendrogram provides a
  hierarchical cluster representation of
  similarities between samples
• SIMILARITY MATRIX The similarity matrix shows
  the percentage of similarity between each pair of
  samples in your report
• OVERLAY The overlay of any pair of isolates can
  be viewed by clicking on the corresponding square
  in the similarity matrix. It provides a graphical
  representation of two samples aligned on top of
  each other.