Kate Templeton
Kate Templeton
Manage acute infection Right treatment Right infection control Right prognosisg p g Promote immunisation Prevent further cases Prevent further cases Prevent likely complications
Right patient Right symptoms Right sample Right transport/shipment/ deliveryg p p y Right test Right report Right report Timely report Right advice Right advice Patient review in light of this report
Respiratory infectionsGastrointestinal
Status of molecular methods
Multitude of techniquesDifferent laboratoriesDifferent samples required Blood culture Sputum Throat swab Throat swab Different media Charcoal Amies Viral transport medium Throat wash/Nasopharygeal aspirate
Clotted blood ( acute and convalescent)
Diagnosis of Respiratory infections
30 minutes30 minutes
15 minutes15 minutes
1 i1 i
2-3 hours2-3 hours
2-3 hours2-3 hours
15 minutes15 minutes
1-2 days1-2 days
1-7 days1-7 days1-21 days1-21 days
8-14 days8-14 days
Conventional Trained/skilled staff
Vi l lt
PCR One extraction
Viral culture 3 cell lines Use shell vial to
Multiple targets In same
Use shell vial to increase sensitivity
IFE h i i
thermocycler 4 way multiplex
Each virus requires specific well
Limited range of
Reduce MMX volume Process lends itself
t t tig
pathogens RSV only good
example
to automation 2 staff
1 ifi ll example
6 staff 1 specifically
qualiifed
Consumable costConsumable cost Total spend and
attribute each cost
200 tests Cultureattribute each cost
to specific test
Test complexity
£22.90/sample
IF( 3 Mab)£21 10/Test complexity
Staff-skill mix required
£21.10/
£8800required
Machine servicesb
PCR 1 extraction
Laboratory spaceOther overheads
4 PCR multiplexs £30
Lighting/ heating/ etc
£6000
Diagnosis of Pneumonia with PCR
Bacterial culture within 1-2 days and PCR within 4 hours.Bacterial culture within 1-2 days and PCR within 4 hours.
Still go furtherStill go further
CaseAdult patient, Renal Tx – increased 02 requirementX-ray changes – suggestive of atypical pneumonia
Blood culture LRT sample
Induced sputum – by physio
5 pots
EDTA bloodCMV PCR
Virology M l TB and atypical bacteria
Bacteria
Virology Mycology TB and atypical bacteriaPCP
CultureIF and PCR
Resp PCR MicroscopyCulture
MicroscopyCulturePCR
CaseAdult patient, Renal Tx – increased 02 requirementX-ray changes – suggestive of atypical pneumonia
Blood culture LRT sample
Induced sputum – by physio
2 pots
EDTA bloodCMV PCR
PCR Bacteria
Resp virus, PCP, MTBAt i l M b t i
CultureAtypical Mycobacteria,Main fungal pathogen
LRT sample for Bacterial CultureClotted blood sample for serology CFT IgM HIV test
Viral throat swab for respiratory viral PCRUrine – Legionella antigen test Blood cultures
Cost - £132 54Cost £132.54
LRT sample Bacterial Culture (£6.74) Viral PCR, Legionella, mycoplasma, Chlamydia
(£35)
Cl tt d bl d Clotted blood HIV (£9.05)
Bl d l (£13 87) Blood cultures (£13.87)
Cost - £64.86
Workload increase in tests
200
250
10- fold increase
150 2004
2012
50
100
0No.
Has it all got out gof control?Or is this a really
l bl i ?valuable service?
AimAimCompare December 2008 to D b 2012 December 2012 Focus on Paediatric A&E attendance
Methods Selected all patients with respiratory sample
taken while in A&E for whole month of D bDecember
Ensure viral investigations complete in both ti i dtime periods
Collect clinical and outcome data
Viruses 2012
2008 Flu A
hRV hMPV Flu A
Flu BRSV
hMPV PIV4 HKU1 Bo detella Pe t ssis RSV
PIV1
HKU1 OC43
NL63
Bordetella PertussisMycoplasma Pneum
PIV2 PIv3
NL63 229E
Adv BoCV
Clinical outcome
Admission ITU/HDU
L h f
Clinical diagnosis
Length of stay Re-admission X-ray
diagnosis ICD coding Bronchiolitis X ray
FBC Blood culture
i l
Pneumonia Viral illness Coryzal illness Urine culture
Antibiotics NG feeding
Coryzal illness LRTI URTI NG feeding
Samples Received
700
625
600
700
400
500
200
300 168
0
100
0
2008 2012
Age profiles
250
300
200
250
1502008
2012
50
100
00 Months 1-3 Months 4-6 Months 7-12 Months 1-2 years > 2 years
How many admitted
2008 2012
Admitted to Hospital
Admission later
HDU/ITU
Admitted to Hospital
Admission later
HDU/ITU
Not admitted Not admitted
Viruses in Bronchiolitis2008 2012
RSV 90 96
hRV 7 55
95% of cases Had viral hRV 7 55
Influenza A 3 7
hMPV 2 3
Had viral diagnosis
hMPV 2 3
Adenovirus 0 2
PIV1 0 3PIV1 0 3
PIV3 0 3
OC43 0 7
BoCV 0 1
No diagnosis 5 5
Total 110 151
Differences 2008
92 patientsAdmitted 80
2012 116 patients
Admitted 62 Admitted 80 Mean LOS 2.1 Xray – 26
Admitted 62 Mean LOS 1.5 Xray- 10Xray 26
Fbc – 21 BC 16
Xray 10 Fbc – 8 BC -10
Antibiotics 16 Antibiotics 6
Costs – 100 patients 2008
H t £126 420
2012
H St £55 650 Hosp stay £126,420 Xray - £916
Hosp Stay - £55,650 Xray - £265
PCR cost £3000 PCR cost £3000 PCR cost - £3000 PCR cost £3000
Overall – there is saving to total budget - £71,724
How to restrict when requiredG d li i l lGood clinical protocols
Good clinical engagement
Robust ITS l h h l l l Simple IT that can match clinical protocols
Stop duplicate testing
Possible future ideas
Costs of tests made more transparentI i t t i t h d b i Increase in tests is matched by service
Gastrointestinal infectionsGastrointestinal infections
Specimen CollectionSpecimen C ll ti
Pre-Analytical Phase
p
Stool container Tx Medium for virus
95% 5%
2nd Floor Accessioning
100%
7th floor Receiving (date & time)
100%
Retreive work: bring to Enteric Lab
100%
Category 3 Room (TB suspected, HIV+, IC)
Identify high risk with tape
Faeces Room (Class 1)
1% 99%
CollectionStool
containerTx Medium for virus
95% 5%
Pre Analtic Phase
2nd Floor Accessioning
100%
7th floor Receiving
(date & time)100%
Retreive work: bring
to Enteric Lab
Retreive work: bring to PCR Lab
100% 100%
Category 3 Room (TB suspected, HIV+, IC)
Identify high risk with tape
Faeces Room (Class 1)
1% 99%0:04:00 0:00:000:04:00 0:00:00
0 911 0 000
Split sample for tests not in GPP, Astrovirus, Sapovirus, Aermonas, Plesimonas and other
OCP
No Split
3% 97%
Appearance; Setup Type & No Media
100%
Emulsify solid specimen
No more prep
1% 99%
Transfer Samples to Testing area
Process xTAG GPP Other Bacteria Other Virus PCR Other OCP
Preparation & ExtractionPreparation &
ExtractionClinical suggested
0.911 0.000Split sample for virology; paperwork
No Split
3% 97%0:02:01 0:00:000:02:01 0:00:00
0.459 0.000Appearance; Label Plates; Setup Type &
No Media100%
0:01:440:44:56
0.395Emulsify solid
specimenNo more prep
1% 99%0:00:51 0:00:000:00:51 0:00:00
0.194 0.000Preparation Media OCP Norovirus Viral Gastro
DecisionCriteria
All stools get XLD plate
All x-Certain Inpatient,i.e., AED, 3X, 3Y,
stoke unit,
All x-Certain Inpatient,i.e., AED, 3X, 3Y,
stoke unit, All 7Y or 10Z
Foreign Travel
Foreign Travel
Vancomycin resistant
enterococci (VRE)
7Y or 10ZAbdominal
pain
Requested On requestClinical
suggested< 14 Y
When ordered
When ordered
> 65 Y outpatient
CONC insufficient
Immunosuppressed
Immunosuppressed
Inpatient suspected
sample Foreign Travel Foreign Travel
17Pathways
Analytialc PhaseObserved batch of 24 samples
Sample Preparation & Extraction. Observed batch 24 tests
Bertin Tube pre-extraction and Qiagen extraction. All stools get XLD plate
Immunosuppressed
Astrovirus Sapovirus Foreign Travel
7Y or 10Z
System set up Prepare and Calibrate MAGPIX System
CONC
Incubate 24hrs at 37C
Run PCR Run PCRConcentration for ova,
cysts and parasites (OCP)
Validate Results Validate ResultsPrepare Faecal Parasite
Concentrator
Prepare Reagents Prepare Master Mix and add to plate
Various parasites
Avg. Wait
If positive, send to Reference Lab & freeze on beads
If positive, send to Reference Lab & freeze on beads
Read ASAP
Examine the slide under the microscope
any clinics any clinics(VRE) suspected
Complex algorithm
5Z, acute GE 7Y or 10Z 7Y or 10Z
Abbrev XLD CAMP SEL - F SMAC SAB APW VIBRIO KANA AESC CN-CIP MAC MAC @ 30 C DIFF Wet Prep CONC CRYPTO Norovirus Viral Gastro
Full Name XLDCampylobacte
r selective agar
Selenite F broth
Sorbitol MacConkey
agar
Sabouraudes agar
Alkaline peptone water
Colorex Vibrio
Chromogenic agar
Gentamicin and
ciprofloxacin discs placed
on MAC
MacConkey agar at 30 degrees C
Clostridium Difficile
Ova, Cycsts, Parasites Direct
Wet Prep
Concentration for ova, cysts and parasites
(OCP)
Stain for Cryptosporidiu
mNoraovirus Viral Gastro
Incubate /1st Step
Incubate 24hrs at 37C
Incubate 48hrs at 42C
(into Bug Box)
Incubate 24hrs at 37C
Incubate 48hr at 37C in air
Incubate 48hr at 37C in air
Incubate APW broth for
24hrs
Incubate 24hrs at 37C
Incubate at 24hrs in air
Incubate at 24hrs in air
Incubate 48hr at 30C
Split into eppendorf tube
2mLPrepare Slide
Prepare Faecal Parasite
ConcentratorPrepare Slide
Preparation & Extraction
Preparation & Extraction
Percent Occurrence 100% 85% 85% 100% Yeasts /
fungus5% 15% Other Other 5% 36% 14% 30% 50% 100% 100%
Labor 0:00:07 0:00:07 0:00:15 0:00:07 0:00:15 0:00:07 0:00:07 0:00:18 0:00:26 0:00:42 0:03:16 0:06:42 0:06:42TAT 0:00:29 0:09:35 0:01:02 0:00:29 0:01:02 0:00:29 0:00:29 0:01:10 0:03:26 0:05:40 0:20:46 1:47:02 1:47:02
ABCost 0.447 0.447 0.538 0.447 0.538 0.447 0.447 0.226 0.147 0.242 1.118 7.556 3.608
Next Step Read at 24 hours
Read at 24 hours
100% Subculture to
XLD incub 24hrs
Read at 24 hours
Incubate Read at 24hrsRead at 24
hours
Make a CRYPTO (volume captured in CRYPTO)
Make a CRYPTO (volume captured in CRYPTO)
Percent Occurrence 100% 100% 100% 100% 100% 100% 100%
Labor 0:00:19 0:00:19 0:00:23 0:00:19 0:00:00 0:00:19 0:00:19TAT 24:00:19 24:00:19 24:00:23 24:00:19 24:00:00 24:00:19 24:00:19
ABCost 0.108 0.108 0.552 0.108 0.108 0.108Reincubate if no growth
Read again at 48h
Read at 24 h
Subculture to APW incubate
Read again at 48h
5 Read at 24 hours
Multiplex PCR and Read
Add samples to plate and run PCR process.
Add Beads and SAPE for hybridisation stage
then read on MAGPIX.
100%
If positive, send to Reference Lab & freeze on beads
Reincubate if no growth 24hrs in airValidate and reportAnalytic Phase
no growth 24hrs in air
48hrs hoursAPW incubate 24hrs at 37C
48hrs
Percent Occurrence 93% 100% 100% 100% 100%
Labor 0:00:26 0:00:19 0:00:19 0:00:15 0:00:19TAT 24:00:26 24:00:19 24:00:19 24:00:15 24:00:19
ABCost 0.149 0.108 0.108 0.568 0.108
Read again at 48hrs
100% Subculture to
VIBRIO (captured in
VIBRIO)100% 100%
0:00:19 Continue w/VIBRIO
24:00:190.108
Continue XLD
P th C l b t Vib i Yersinia Cl t idi V i V i C t idi
Pathways
Positive C Difficile samples reflexed for Toxin EIA as per Governement directive.
Bacterial positive samples typed for epidemiology.
Aeromonas Plesiomonas
Pink colonies Pink colonies
Oxidase positive Oxidase positive
Confirm MALDITOF Confirm MALDITOF
O-129 on blood agar plate, incubate
Sensitivity on isosensitest agar -against gram ring
Send to NTL, freeze on Send to NTL, freeze on
Pathogen Type Aeromonas Campylobact
er Plesiomonas E. Coli O157 Salmonella Shigella Vibrio Species Enterocolitic
a
Clostridium Difficile
Various parasites
Various parasites
Cryptosporidium Noravirus Adenovirus Astrovirus Rotavirus Sapovirus
Positive if Pink coloniesCAMP will have grey colonies
Pink coloniesSMAC - grey
coloniesBlack center
colonies
Pink colonies, no black centers
Pale blue, muave
colonies
Small non lactose
fermenterAvg. Wait Avg. Wait Avg. Wait Avg. Wait Avg. Wait Average Wait
Oxidase Oxidase positive
Oxidase positive
Oxidase positive
Oxidase negative
Oxidase negative
Oxidase negative
Oxidase positive
Oxidase negative
Percent Suspect 1% 6% 1% 19% 5% 3% 3% 2% Read ASAP
Read every 2-3 hrs
Read once per day
Determine +/- 0:03:38 0:03:19 0:03:38 0:03:19 0:03:19 0:03:19 0:03:19 0:03:19
TAT 0:05:13 0:04:54 0:05:13 0:04:54 0:06:38 0:06:38 0:06:38 0:04:54ABCost 1.243 1.135 1.243 1.135 1.135 1.135 1.135 1.135
Next Step(s) Confirm MALDITOF
Confirm MALDITOF
Confirm MALDITOF
Sub-culture suspect
colonies to blood agar overnight
Confirm MALDITOF
Confirm MALDITOF
Confirm VITEK
Confirm MALDITOF
Perform Kit TestExamine the
slide under the microscope
Examine the slide under the
microscope
Examine under UV Microscope
Run PCR Run PCR Run PCR Run PCR Run PCR
Percent 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%Labor 0:09:00 0:09:00 0:09:00 0:00:36 0:09:00 0:09:00 0:18:01 0:09:00 0:22:16 0:02:30 0:03:47 0:01:11 0:12:05 0:12:05 0:12:05 0:12:05 0:12:05TAT 0:11:00 0:11:00 0:11:00 24:00:36 0:11:00 0:11:00 15:54:01 0:11:00 0:55:00 0:04:59 0:18:55 0:09:31 4:39:57 4:39:57 4:39:57 4:39:57 4:39:57
ABCost 4.079 4.079 4.079 0.480 4.079 4.079 8.664 4.079 5.164 0.853 1.294 0.407 12.570 6.245 6.245 6.245 6.245
O-129 on blood agar
plate, incubate
BSENS Sensitivity overnite
24hrs at 42C
Sensitivity on isosensitest
agar - against gram ring
Test non sorbitol
fermenters (NSF) w/latex
kit
Agglutinating Sera Test
Agglutinating Sera Test
Subculture to Blood Agar
purity Incubate
24hrs at 42C
Sensitivity on isosensitest
agar - against gram ring
If positive, send to Reference
Lab & freeze on beads
If positive, send to Reference
Lab & freeze on beads
If positive, send to Reference
Lab & freeze on beads
Positives perform
Modified Ziehl Neelsen (ZN)
If positive, send to
Reference Lab & freeze
on beads
If positive, send to
Reference Lab & freeze
on beads
If positive, send to
Reference Lab & freeze
on beads
If positive, send to
Reference Lab & freeze
on beads
If positive, send to
Reference Lab & freeze
on beadsPercent 1% 83% 1% 100% 0.5% 0.2% 70% 0.4% 100% 0.3% 0.3% 1% 21% 5.3% 2.4% 3.4% 1.2%
30
beads, final report beads, final reportLabor 0:01:51 0:00:55 0:01:51 0:13:45 0:06:27 0:06:27 0:00:07 0:01:51 0:20:00 0:20:00 0:20:00 0:01:31 0:20:00 0:20:00 0:20:00 0:20:00 0:20:00TAT 24:01:51 0:02:55 24:01:51 0:43:45 0:06:27 0:06:27 0:00:07 24:01:51 0:20:00 0:20:00 0:20:00 0:41:31 0:20:00 0:20:00 0:20:00 0:20:00 0:20:00
ABCost 1.055 0.589 1.055 4.979 2.457 2.457 0.461 0.910 31.842 31.842 31.842 0.521 31.842 31.842 31.842 31.842 31.842
Send to NTL, freeze on
beads, final report
Subculture to Blood Agar
purity Incubate
24hrs at 42C
Send to NTL, freeze on
beads, final report
VITEK 2 (to distinquish Shigella)
Sensitivity on isosensitest
agar + Nalaixic Acid
Sensitivity on isosensitest
agar + Nalaixic Acid
Sensitivity on isosensitest
agar - against gram ring
Send to NTL, freeze on
beads, final report
If positive, send to Reference
Lab & freeze on beads
Percent 100% 50% 100% 100% 90% 90% 100% 100% 100%Labor 0:23:07 0:00:36 0:23:07 0:18:01 0:01:51 0:01:51 0:01:51 0:23:07 0:20:00TAT 0:23:07 24:00:36 0:23:07 15:54:01 24:01:51 24:01:51 0:01:51 0:23:07 0:20:00
ABCost 32.907 0.480 32.907 8.664 1.185 1.185 0.910 32.907 31.842Send to NTL,
freeze on beads, final
report
Sensitivity on isosensitest
agar
If Naladixic resistent, test
CiproEtest
If Naladixic resistent, test
CiproEtest
Send to NTL, freeze on
beads, final report
Percent 100% 13% 50% 5% 100%Labor 0:23:07 0:00:55 0:00:36 0:00:36 0:23:07TAT 0:23:07 24:00:55 24:00:00 24:00:00 0:23:07
ABCost 32.907 0.589 0.480 0.480 32.907If positive,
send to Reference
Lab & freeze on beads
If positive, send to
Reference Lab & freeze
on beads
If positive, send to
Reference Lab & freeze
on beads100% 100% 100%
0:23:07 0:20:00 0:20:000:23:07 0:20:00 0:20:00
Luminex wins $11 6Mmilitary contractLuminex wins $11.6M military contract
Positive negative and coPositive, negative and co‐infection results reported at the same time with GPP.
31
Luminex wins $11 6Mmilitary contract6 Months LIS
Luminex wins $11.6M military contract data showed a potential to reduce samples by 45%
32
Summary Need to look at each step of the process Need to look at each step of the process Often the system is very complicated
Historical Multiple labs Multiple tests IT IT Transport
PCR automation has enabled less staff to do considerably more testsconsiderably more tests
Multiplex approach – can enable cost neutral introduction
Perform time-in motion and detailed cost analysis Ensure the range of pathogen possibilities are
covered – link protocols within different laboratoriesp
Future Better link with the clinical need Enable budgets to move to deflect change in
clinical practiceMake tests than results in patient pathway Predictive – e.g genetic or host susceptible
markers
Continue to focus on overall picture Not individual tests
Use of models