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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 problemMicrobiological 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 : 1992 - 2000
•1.34 million cases in 2000
•>325,000 general practitioner consultations
•20,800 hospital admissions
•> 88000 bed days
•480 deaths
FSA Research and Survey Programmes Annual Report 2007
Food-borne Illness Health Risks:Numbers Affected and Severity (2000)
Pathogens Deaths Hospitalisations
All Cases
Total Number
Ranking Total Number
Ranking Total Number
Ranking
Campylobacter spp
90 2nd 16,950 1st 359,500 1st
E.Coli O157 20 380 3rd 1,000 3rd
Listeria monocytogenes
70 3rd 190 200
Salmonellas non-typhoidal
120 1st 1,520 2nd 41,600 2nd
Food-borne Illness Health Risks:Numbers Affected and Severity (2005)
Pathogens Deaths Hospitalisations
All Cases
Total Number
Ranking Total Number
Ranking Total Number
Ranking
Campylobacter spp
70 3nd 13,930 1st 295,500 1st
E.Coli O157 20 400 3rd 1,100 3rd
Listeria monocytogenes
130 1st 380 400
Salmonellas non-typhoidal
100 2ndt 1,220 2nd 33,400 2nd
Laboratory Reporting of Selected GI Pathogens in England & Wales.
0
10
20
30
40
50
60
77 79 81 83 85 87 89 91 93 95 97 9920
01
Year
Lab
rep
ort
s (1
000'
s)
Cryptosporidium
Rotavirus
Campylobacters
Salmonellas
Shigellas
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
The problemMicrobiological 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 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)
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
MDEP Molecular detection of entericpathogens 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/coliGiardia, 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.
Run Date: 30.10.07 Machine ID: CSB2 2.16PCR Target: ABI Trial Plate Operator: LN MR Saved As: 30.10.07 Trial Plate 2 Master mix Batch: Environmental
Ecoli
0157
VT2 +
veOrganism
Salm
onel
la +
ve
Salm
onel
la +
ve
C.jeju
ni +
ve
+ve co
ntro
ls
Crypt
o +ve
Crypt
o +ve
Giard
ia +
ve
Giard
ia +
ve
C.jeju
ni +
ve
C.jeju
ni +
ve
Ecoli
0157
VT1 & V
T2
+ve
Lab numberM07.404071 M07.406856 M07.403281 M07.403360 M07.403862 P07.405928 P07.407083 P07.408731 M07.404188 P07.406566 P07.406404
Wet assay CT
17.73 31.88 23.16 38.97 29.82 34.4 /34.75/ NT
45/27.76/ NT
35.1 31.35 29.36 32.6
Assay 1 2 3 4 5 6 7 8 9 10 11 12A Crypto
30.48 28.35VT1 +ve control
B Giardia
New extract44.41
New extract
VT2 +ve control
C C. coli
Salmonella +ve control
D C. jejuni
21.26 New extract 27.26C.coli +ve
control
E Salmonella
17.01 32.00C.jejuni
+ve control
F VT 1
32.03Giardia +ve
control
G VT2
33.17 26.11Crypto +ve
control
H E.coli 0157 IPC
45 E.coli 28.16 IPC
38.31 E.coli 26.69 IPC
38.83 E.coli 26.78 IPC
41.38 E.coli 27.29 IPC
40.51 E.coli 27.35 IPC
30.63 E.coli
27.05 IPC
26.33 E.coli 26.85 IPC
45 E.coli 27.5 IPC
43.34 E.coli 27.22 IPC
17.99 E.coli 29.21 IPC
45 E.coli 27.05 IPC
E.coli +ve control
Discrepant Result Inhibitory
Ecoli
0157
VT2 +
veOrganism
Salm
onel
la +
ve
Salm
onel
la +
ve
C.jeju
ni +
ve
** diluted 1:100 NT - Not tested
+ve co
ntro
ls
Crypt
o +ve
Crypt
o +ve
Giard
ia +
ve
Giard
ia +
ve
C.jeju
ni +
ve
C.jeju
ni +
ve
Ecoli
0157
VT1 & V
T2
+ve
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 48-72 hr
Real time study-approx 2 months, 612 faecal specimens Community and HospitalDiscrepant analysis4 Salmonella21 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