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 : 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
Hospitalis
ations
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
Salmonellas
non-typhoidal
120
1st
1,520
200
2nd
41,600
2nd
Food-borne Illness Health Risks:Numbers
Affected and Severity (2005)
Pathogens
Deaths
Hospitalis
ations
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
Salmonellas
non-typhoidal
100
2ndt
1,220
400
2nd
33,400
2nd
Laboratory Reporting of Selected
GI Pathogens in England & Wales.
Cryptosporidium
Rotavirus
Campylobacters
Salmonellas
Shigellas
50
40
30
20
10
Year
20
01
99
97
95
93
91
89
87
85
83
81
79
0
77
Lab reports (1000's)
60
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 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 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
PCR assay development
Real time amplification and detection evaluated
Lightcycler
TaqMan
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
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VT i 0 1
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Sa
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Organism
Ec
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VT i 0 1
1 57
+v & V
e T2
Machine ID: CSB2 2.16
Operator: LN MR
Master mix Batch: Environmental
30.10.07
Run Date:
ABI Trial Plate
PCR Target:
Saved As: 30.10.07 Trial Plate 2
M07.404071 M07.406856 M07.403281 M07.403360 M07.403862 P07.405928 P07.407083 P07.408731 M07.404188 P07.406566 P07.406404
Lab number
Wet assay
CT
Assay
A
Crypto
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
1
2
3
4
5
6
7
8
9
10
11
30.48
28.35
12
VT1 +ve
control
New extract
44.41
New extract
VT2 +ve
control
B
Giardia
Salmonella
+ve control
C
C. coli
D
C. jejuni
E
Salmonella
21.26
17.01
New extract
C.coli +ve
control
27.26
C.jejuni
+ve control
32.00
Giardia +ve
control
F
VT 1
32.03
G
VT2
33.17
26.11
30.63 E.coli
27.05 IPC
26.33 E.coli
26.85 IPC
H
E.coli 0157
IPC
45 E.coli
28.16 IPC
** diluted 1:100
38.31 E.coli 38.83 E.coli 41.38 E.coli 40.51 E.coli
27.35 IPC
27.29 IPC
26.78 IPC
26.69 IPC
Discrepant Result
Inhibitory
Crypto +ve
control
45 E.coli
27.5 IPC
43.34 E.coli 17.99 E.coli
29.21 IPC
27.22 IPC
NT - Not tested
45 E.coli
27.05 IPC
E.coli +ve
control
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 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
Manchester
Preston
Lynne Newbold
Caroline Durband and
Mark Regan
colleagues
Gemma (MSc)
Bernard Wood/Malcolm Armstrong
Enteric Staff
MRI/Wythenshawe