Enteric PCR: test
evaluations and
application of PCR in
clinical microbiology
Kate Templeton
NHS Lothian
1
WHO data
2
Workload in Edinburgh, UK
3
Positives in Scotland
Pathogen
2013
2014
Norovirus
1915
1306
Rotavirus
1301
346
Shigella sonnei
62
49
Shigella flexneri
19
34
Yersinia entercolitica
6
4
Campylobacter
6163
5293*
Listeria
15
15
C.difficile
1731
1710
Salmonella
813
543*
Cryptosporidium
429
VTEC
219
Data HPS
282*
4
* Reported at week 40
Service challenges we face

Increased workload

Less staff

Skilled staff retiring

Less money

improve clinical service

Equity of access

Patient centred care

Fast and accurate results
5
Current enteric
pathogen
processing
6
Overlapping process
Norovirus
•Hospitalised/outbreaks in community
C.diffiicle
•90% of above plus
any person >15 with
diarrhoea
Enteric
culture
Crypto
•All tested for C.diff
plus others with
signs of
gastroenteritis
Microscopy
A subset of those
getting culture, e.g
travel,
immunocompromsied
7
Patient presents in Hospital with
acute diarrhoea – what most
likely

Norovirus

Campylobacter

C.difficle

VTEC

Shigella

Salmonella


Combine NoV and C.diff
Could be any of them !
Stool only requested for both C.diff and Norovirus – 30%
of time.
8
C.Difficle Challenges
Screen with GDH or PCR ( toxin b)
Confirm with toxin test
Screening with PCR is an option
9
C.Difficile Challenges
10.0%
9.0%
Equivocals
8.0%
48/95
- PCR neg
7.0%
1.75 visits/ case for IPCN
6.0% moved to side room
45%
5.0%
4.0%
% equiv
% Pos
3.0%
2.0%
1.0%
0.0%
10
Parasites – what is best method for diagnosis?
Cyst of Giardia lamblia
Cyst of Entamoeba histolytica
Cyst of Cryptosporidium parvum
Reference: Resources in Medical Microbiology. Scion Publishing Ltd. Viewed 24th September 2013 at
<http://www.scionpublishing.com/shop/collection_display.asp?currencyid=2&collectionid=%400000000248>
Parasite PCR
PCR is gold standard
Microscopy 30-50% sensitive 1,2
12
1 van Lint 2014 et al
2 Stark 2011 et al
PCR as an option
Norovirus
PCR
•Hospitalised/outbreaks in community
C.Difficile
PCR
•90% of above
plus any person
>15 with
diarrhoea
Enteric
culture
VTEC
Crypto PCR
•All tested for
C.diff plus
others with
signs of
gastroenteritis
Parasites
PCR
13
Can we do this with less
staff and same money?
14
Norovirus on BDMax

BDMAX is an
Integrated
extraction and PCR
platform

Evaluated in 2012
for Norovirus testing
Introduced in 2013
The BDMAX

Extraction on URS

PCR on cartridge
Workflow
1mL H2O +
25MG SAMPLE
SAMPLE
PREP
EXTRACT
1mL H2O +
25MG SAMPLE *SBT
SAMPLE
PREP
PCR
**URS
EXTRACT
ANALYSE
PCR CARTRIDGE
PCR
ANALYSE
*Sample Buffer Tube
**Universal Reagent Strip
Validation Results
RESULTS
Platform
TP TN
FP FN Sensitivity
ABi IC
99
233 2
3
BDMAX
IC
96
233 2
6
Specificity
PPV
(%)
NPV
(%)
97%
(91.0-99.2)
99%
(96.6-99.8)
98
(92.3-99.7)
99
(96.0-99.7)
94%
(87.1-97.6)
99%
(96.6-99.8)
98
(92.1-99.6)
98
(94.4-98.9)
ASSAY COMPARISON
P Value
Cohen’s Κ
easyMAG/ABi NO IC vs. easyMAG/ABi 7500 IC
1.00
0.96
BDMAX IC vs. easyMAG/ABi NO IC
0.67
0.89
easyMAG/ABi 7500 IC vs. BDMAX IC
0.80
0.94
Outcomes for the service

BDMAX requires less technical skill and
hands on time to perform the test and
analyse results

BDMAX currently being run by band 2s and 4s

Lab TAT – 4 hours 17 mins

Compared to Cepheid -3 hours 50 mins

70-80% of results received by IPCN by
15.00

Decrease in lost beds


2007-9 3678 beds closed due to Norovirus –
Daniel et al 2011

20013-15 253 beds closed due to Norovirus
7 days testing Weekend testing informs
ward closures at weekend
Multiplex with C.difficile
for same staff
Cost of GDH - £6 per test
Current consumable cost =£108,000
If combined PCR – get 7000 test( no additional charge – as already doing NoV)
Additional PCR cost =£110,000
Advantages – No missed diagnosis
Staff time overall saving
20
Now for the rest!!
21
22
Validation
Sample set:
• 412 Stool samples
• 189 – from viral lab
• 233 – from bacteria enterics lab and E.coli ref
lab
• Process with GPP panel and compare to
existing routine (and new) in-house real-time
PCR as well as culture
189 Virology Samples
Noro
Rota
Adeno
Neg
PCR
53
13
13
110
GPP
50
11
2
67
Missed by
GPP
10 (ct>35)
2 (ct>38)
11 (ct14-40)
Sensitivity
90%
94%
20%
Additional positives
• 6 C. Diff
•3 Salmonella
•32 Camplyobacter
•2 Crypto
Numerous samples with > 1 pathogen
25 had Bacteria and virus
1 Noro, Rota, Camplyobacter and C.diff
Bacteria Enterics Samples
GPP
Culture
Missed by GPP
Salmonella
24
32
12
Shigella
9
4
0
Camplyobacter
45
39
1
C.difficile
24
23
1
ETEC
8
5
0
STEC
7
8
3
O157
7
10 (Inc IMS)
3
Yersinia
enterocolitica
0
0
0
Vibrio chloera
0
1
1
Parasite Enteric Samples
Microscopy
GPP
PCR
Missed By
GPP
Giardia
4
6
6
0
Cryptosporidium
12
12
13
1
Entamoeba
histolytica
0
0
0
0
Additional Positives
•
•
•
•
10 Norovirus positives – in Enteric samples
29 Dual Infections
5 Triple infections
1 with 4.
Got Local grant to assess PCR
for bacteria/parasites

Awarded £39K from the ELHF

http://www.elhf.co.uk/
28
Phase One: Retrospective Testing
BDMAX ENTERIC PANELS
ENTERICBIO GASTRO PANEL 2
CULTURE AND MICROSCOPY
LUMINEX XTAG GPP
INHOUSE ASSAYS
29
Quick Assay Comparison
BDMAX
Enteric Bacterial Panel
Plus
Enteric Parasite Panel
Salmonella Spp.
Campylobacter jejuni/coli
Shigella spp/EIEC
VTEC Stx1/Stx2
Shigella dysenteriae.
Giardia lamblia
C. hominis/parvum
Entamboeba histolytica
24 Samples per run
Luminex
xTAG® GPP
AdV 40/41,
NoV GI/II,
Rota A
C. diff toxin A/B,
Campylobacter spp.,
E. coli O157,
ETEC HL/HS ET,
VTEC(Stx)1/2,
Shigella spp.,
Salmonella spp.,
V. cholerae,
Y. enterocolitica,
Cryptosporidium spp.,
Giardia spp.
E.histolytica
96 Samples per run*
Serosep
EntericBio realtime
Gastro Panel 2
Salmonella Spp.
Campylobacter jejuni/coli/lari
Shigella spp/EIEC
VTEC (stx1/stx2)
Shigella dysenteriae
C. parvum/hominis
Giardia lamblia
46 Samples per run
Sample to Answer: ~3Hrs Sample to Answer: 4.5 Hrs Sample to Answer:<3Hrs
Hands-on-time: 30mins
Hands-on-time: 1.75 Hrs
Hands-on-time: 30mins
Low Complexity
High Complexity
Low Complexity
£20.82 per assay
Plus external controls
£75 plus £4 extraction
Via easyMag
30
~ £12.50
PANEL

Salmonella

Shigella

VTEC

Camplyobacter

Giardia

Crytosproridium

50 negatives

Positives collected over 1 year period

Approx 20 -30 of each
31
POSITIVES
BDMAX
SEROSEP
0
0
108
22
2
19
CULTURE/MICROSCOPY
THIS EXCLUDES AND UNRESOLVED, INDETERMINATE OR DUAL INFEC
SALMONELLA
BDMAX
SEROSEP
1
0
0
8
1
0
3
CULTURE/MICROSCOPY
CAMPYLOBACTER
BDMAX
SEROSEP
3
0
0
36
11
4
10
CULTURE/MICROSCOPY
CRYPTOSPORIDIUM
BDMAX
SEROSEP
0
0
0
9
2
2
1
CULTURE/MICROSCOPY
VTEC
BDMAX
SEROSEP
1
0
2
35
3
7
13
CULTURE/MICROSCOPY
Dual Infections
ENT
#
C&M
BDMAX
SEROSEP
COMMENT
10
Salmonell
a POS
Salmonella and
VTEC POS.
Salmonella and Looks genuine.
VTEC POS.
69
Salmonell
a POS
Salmonella
POS.
Salmonella POS This could be a possible contamination
and Campy
on the Serosep plate as there are a lot
POS.
of campys on the run.
107
Campy
POS
Shigella POS
and Campy
POS.
Shigella POS
and Campy
POS.
This does look genuine but there is
another strong Shigella positive close
in the run.
201
Salmonell
a POS
Salmonella POS
and VTEC POS.
VTEC POS.
The VTEC result looks genuine but is at
a much lower level than the
Salmonella POS.
208
Campy
POS
Shigella POS
and Campy
POS.
Shigella POS
and Campy
POS.
I think this looks genuine for the
additional Shigella POS.
230
Campy
POS
Shigella and
Campy POS.
NEGATIVE.
There are two strong Shigella positives
either side of this sample so this could
be likely contamination.
Hands on time
STEP SEROSEP PROTOCOL
TIME PER RUN
Innoculate SPS
00:20
Boil SPS
00:30
Set up workstation
00:03
Robotic dispensing
00:40
Mix
00:01
Centrifuge
00:01
Real Time PCR
01:22
Analysis
00:10
HANDS ON TIME
00:35
TIME TO RESULT
03:07
38
Overall
Xtag
BDMax GPP
SersoSep
Inhouse
Set-up time
01:35
00:55
03:30
02:30
PCR
01:22
02:42
04:38
01:40
Hands on time
00:35
00:30
03:38
00:50
Time to result
03:32
04:07
08:09
05:00
46
24
96
46
batch size
39
Next phase -
Test in parallel one system for 2 months with culture
methods

Report out in parallel in LIMS system

Act on results

Support from IPCN

Public health

Protocols developed
40
Summary

In evaluations – need to address clinical problem

Use of automation – it is only solution

All systems look to perform satisfactory in retrospective
testing

Cost

Making case within own resources are main reasons for
chance of success

Be imaginative !!
41
Acknowledgments

Microbiology – molecular Microbiology

Dr. Juliet Kenicer

Dr. Lesley Allison and SERL

Dr. Mary Hanson

Dr. Richard Othieno

Laura McKenzie

Julie White

ELHF for providing funding for project

NES for funding clinical science traineeship
42
Any Questions?