Kate Templeton - smvn.scot.nhs.uk

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


1 i1 i

2-3 hours2-3 hours

2-3 hours2-3 hours


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


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



(into Bug Box)


Incubate 48hr at 37C in air

Incubate 48hr at 37C in air

Incubate APW broth for

24hrs


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%


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



If positive, send to Reference

Lab & freeze on beads





Positives perform

Modified Ziehl Neelsen (ZN)

If positive, send to

Reference Lab & freeze

on beads



on beads



on beads



on beads



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


beads, final report

VITEK 2 (to distinquish Shigella)


agar + Nalaixic Acid


agar + Nalaixic Acid




beads, final report



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


agar

If Naladixic resistent, test

CiproEtest

If Naladixic resistent, test

CiproEtest


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




on beads



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

Documents

Kate Templeton - smvn.scot.nhs.uk