Tomorrow’s world-molecular detection of enteric pathogens Prof Andrew Fox Regional HPA Laboratory/FEMS North West

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

The problem Microbiological causes of diarrhoea: viral bacterial parasitological toxin Range of different methodologies used microscopy culture immunoassay……… Usually <50% of cases have an identified aetiology Important to identify the aetiological agent for appropriate treatment, interventions and control

Foodborne Disease in England and Wales : million cases in 2000 >325,000 general practitioner consultations 20,800 hospital admissions > bed days 480 deaths FSA Research and Survey Programmes Annual Report 2007

Food-borne Illness Health Risks:Numbers Affected and Severity (2000) PathogensDeathsHospitalis ations All Cases Total Number RankingTotal Number RankingTotal Number Ranking Campylobacter spp 902 nd 16,9501 st 359,5001 st E.Coli O rd 1,0003 rd Listeria monocytogenes 703 rd Salmonellas non-typhoidal 1201 st 1,5202 nd 41,6002nd

Food-borne Illness Health Risks:Numbers Affected and Severity (2005) PathogensDeathsHospitalis ations All Cases Total Number RankingTotal Number RankingTotal Number Ranking Campylobacter spp 703 nd 13,9301 st 295,5001 st E.Coli O rd 1,1003 rd Listeria monocytogenes 1301st Salmonellas non-typhoidal 1002nd t 1,2202 nd 33,4002nd

Laboratory Reporting of Selected GI Pathogens in England & Wales.

Infectious intestinal disease study, England 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

The problem Microbiological causes of diarrhoea: viral bacterial parasitological toxin Range of different methodologies used microscopy culture immunoassay……… Usually <50% of cases have an identified aetiology Important to identify the aetiological agent for appropriate treatment, interventions and control

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 /100,000 Incidence of Giardiasis E and W /100,000 Central Lancs incidence of Giardia /100,000 (6 times national rate)

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!

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

PCR assay development Real time amplification and detection evaluated LightcyclerTaqMan

MDEP Molecular detection of enteric pathogens for the routine diagnosis of gastrointestinal infections-HPA modernisation fund

Potential Advantages of Molecular Detection of Enteric pathogens Improved sensitivity Speed of detection Processivity

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

Project four phases 1.Identify universal extraction protocol 2.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

Project phases 3.“Real time” study-processivity 4.Alternative platforms-discrepant analysis Line probe Loopamp

Target Pathogens Campylobacter jejuni Campylobacter coli Salmonella Cryptosporidium VT 1 VT2 O157 IPC

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

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)

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.

PCR assay Real time amplification and detection evaluated TaqMan

Assay Format

Turn around time PCR 90% of specimens results available within <24 hours majority same day-weekend effect Rate limiting steps-machine capacity (fast assays) Extended working day Culture 90% specimens results available for reporting hr

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)

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.

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

Conclusions cont Real time epidemiology Surveillance Information for action

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

Acknowledgements ManchesterPreston Lynne NewboldCaroline Durband and Mark Regancolleagues Gemma (MSc) Bernard Wood/Malcolm Armstrong Enteric Staff MRI/Wythenshawe