Tomorrows worldmolecular detection of enteric pathogens

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Tomorrows world-molecular detection of enteric
pathogens

• Prof Andrew Fox
• Regional HPA Laboratory/FEMS North West

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The problem

• In the UK
• 1 in 5 members of the population are
• affected by food poisoning each year
• 9.4 million in England
• Estimated cost
• 0.75 billion
• 55 to employers
• 36 to health service
• 8 directly to the case

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The problem

• Microbiological causes of diarrhoea
• viral
• bacterial
• parasitological
• toxin
• Range of different methodologies used
• microscopy
• culture
• immunoassay
• Usually lt50 of cases have an
• identified aetiology
• Important to identify the aetiological
• agent for appropriate treatment,
• interventions and control

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Foodborne Disease in England and Wales 1992 -
2000

• 1.34 million cases in 2000
• gt325,000 general practitioner consultations
• 20,800 hospital admissions
• gt 88000 bed days
• 480 deaths

FSA Research and Survey Programmes Annual Report
2007
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Food-borne Illness Health RisksNumbers Affected
and Severity (2000)
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Food-borne Illness Health RisksNumbers Affected
and Severity (2005)
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Laboratory Reporting of Selected GI Pathogens in
England Wales.
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Infectious intestinal disease study, England
1993-6

• Case control study to identify the
  micro-organisms and toxins in stool specimens
  associated with infectious intestinal disease
  among cases in the community and presenting to
  GPs and in asymptomatic controls
• 3654 cases
• 2819 controls

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The problem

• Microbiological causes of diarrhoea
• viral
• bacterial
• parasitological
• toxin
• Range of different methodologies used
• microscopy
• culture
• immunoassay
• Usually lt50 of cases have an
• identified aetiology
• Important to identify the aetiological
• agent for appropriate treatment,
• interventions and control

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Choice of method impacts on ascertainment

• Introduction of EIA (RPH Microbiology) in 2002 on
  detection of Giardia in Central Lancashire
• Central Lancs incidence Giardia in 2002
  10.1/100,000
• Incidence of Giardiasis E and W 2005 5.5/100,000
• Central Lancs incidence of Giardia 2006
  33.6/100,000 (6 times national rate)

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Techniques and technologies

• Generic techniques
• Immunoassays
• PCR
• Nucleic acid extraction
• Automated nucleic acid extraction
• Conventional PCR
• Real time PCR
• Other detection techniques
• Robotics

Need to be all singing and dancing!
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Generic nucleic acid extraction developed

• Faeces contains PCR inhibitors
• Methods developed applicable
• to extraction of nucleic acid from
• Viruses
• Bacteria
• Parasites

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PCR assay development

• Real time amplification and detection evaluated
• Lightcycler TaqMan

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MDEP Molecular detection of entericpathogens for
the routine diagnosis of gastrointestinal
infections-HPA modernisation fund
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Potential Advantages of Molecular Detection of
Enteric pathogens

• Improved sensitivity
• Speed of detection
• Processivity

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• Improved turn around time
• Automation and staffing
• Molecular epidemiology
• Inform Pathology Modernisation Agenda

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Project four phases

• Identify universal extraction protocol
• Assay selection
• Target Pathogen assays
• In house (Campylobacter jejuni/coli Giardia
  , Cryptosporidium),
• commercial (VTEC VT1 and VT2, O157)
• Format- wet assays, in plate
• Validation-culture positive specimens

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Project phases

• Real time study-processivity
• Alternative platforms-discrepant analysis
• Line probe
• Loopamp

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Target Pathogens

• Campylobacter jejuni
• Campylobacter coli
• Salmonella
• Cryptosporidium
• VT 1
• VT2
• O157
• IPC

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easyMAG Extraction

• easyMAG setup
• (5 minutes)
• Switch easyMAG on, wait for the orange light to
  turn green then turn computer on.
• Log on
• Barcode reagents Lysis Buffer, Extraction
  Buffer1, Extraction Buffer 2, Extraction Buffer 3
  Magnetic silica beads

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easyMAG Extraction

• OFF Board Lysis
• Place rotor on the MagNA Lyser shaft.
• Put the retention plate on top the rotor, rotate
  it into the closed position and tighten red
  discs.
• Close Lid
• Set the speed to 3000 rpm and the time to 60
  seconds. Press START on MAGNA Lyser.
• (2minutes)

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TAQMAN

• Preparation of Mastermix
• (7 minutes)
• Take out reagents from freezer x2 universal
  mastermix, primers probes.
• Make up mastermix according to the protocol.
• Dispense 20µl into each well in use.

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PCR assay

• Real time amplification and detection evaluated
• TaqMan

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Assay Format
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Turn around time

• PCR
• 90 of specimens results available within lt24
  hours majority same day-weekend effect
• Rate limiting steps-machine capacity (fast
  assays)
• Extended working day
• Culture
• 90 specimens results available for reporting
  48-72 hr

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Real time study-approx 2 months, 612 faecal
specimens Community and Hospital Discrepant
analysis 4 Salmonella 21 Campylobacter spp
(C.jejuni 15, C.coli 5, both 1)
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Fig 2. Normalised melt curves of adk 12 assay.
The melt profiles shown are for an isolate with
adk 12 (SNP T) and nine isolates with different
alleles.
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Conclusions

• PCR increased sensitivity/specificity
• Universal extraction RNA viruses to
  Cryptosporidia
• Multiple target testing-improved disease
  ascertainment
• Improved turn around times
• Modernisation
• Improved public health management of GI
  infections

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Conclusions cont

• Real time epidemiology
• Surveillance
• Information for action

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Molecular detection will considerably improved
the diagnostic gap for detection potential
pathogens
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Acknowledgements

• Manchester Preston
• Lynne Newbold Caroline Durband and
• Mark Regan colleagues
• Gemma (MSc)
• Bernard Wood/Malcolm Armstrong
• Enteric Staff
• MRI/Wythenshawe