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

PCR assay development

Real time amplification and detection evaluated

Lightcycler TaqMan

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.

PCR assay

Real time amplification and detection evaluated

TaqMan

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

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