Viral Gastroenteritis –

patient management and the need for rapid

diagnostics

Susan Feeney

Regional Virus Lab, BHSCT

September 2017

Viral gastroenteritis

5 significant

enteric

pathogens

1. Faecal Adenovirus family Adenoviridae

Serogroups 40 41

Non-enveloped DNA virus

Infant association

Nosocomial associations in CH Haematology

2. Astrovirus , family Astroviridae

Non-enveloped RNA virus

May be asymptomatic

Infant association

90% children antibody by age 9

3. Sapovirus, family Caliciviridae

Non-enveloped RNA virus

Mild gastroenteritis

Infant association

4. Rotavirus, family Reoviridae

dsRNA non-enveloped, segmented virus

>90% antibody positive by age 5

Sept 2013 Rotarix Vaccine UK

Oral vaccine at 8 and 12 weeks

85% efficacy

2014, 2015, 2016 reported cases

fell by 70%

5. Norovirus, family Caliciviridae

RNA non-enveloped virus

Most common cause gastroenteritis

Affects all ages

Associated with outbreaks in closed

Or semi-closed environments

5 Genogroups , GI GII human associated

Diagnostics

EM

ELISA

IF

Virology Enterics:

Taqman™ real time PCR in-house (LDT) Norovirus/IC; Rotavirus/IC, Adenovirus/Astrovirus/IC.

Approx 4000 faeces/vomit specimens per year

Seasonal distribution of Norovirus GI/GII positive

specimens in both paediatric (Yellow) and adult (Blue)

population over a three year period March 2014-February

2016.

Paediatric yellow

Adult Blue

‘Winter Vomiting Bug’

EntericBio Realtime Norovirus Assay

The Serosep EntericBio Norovirus real time assay is a recently released semi-automated, CE-marked assay.

Taqman™ PCR assay for the qualitative detection of NoVs genogroup I and II in faecal specimens.

Employs a novel system for liberation of nucleic acid directly from faecal samples and does not require nucleic acid extraction and purification steps.

PCR assays are internally controlled and set-up is from pre-dispensed, lyophilized mastermix test strips.

This system offers speed (46 specimens <3hrs), minimal hands-on and LIMs interfacing to ensure optimum turn-around with patient safety and result reliability as priority.

Assay Steps

Faecal sample is re-suspended in EntericBio realtime SPS solution

Heat Treat 103°C (30 min)

Centrifuge (1 min)

No Extraction! No associated Instrumentation.. No associated Maintenance.. No associated Consumables.. No associated Reagents.. No Time lost on extraction run!

Shake (1 min)

Centrifuge (1 min)

PCR Set-up:

Eppendorf transfer of faeces suspension to PCR

strips containing predispensed mastermix (30 min)

LightCycler 480 (Roche) PCR (1 hr)

Automated EnterBio Realtime macro analysis.

Multicentre Clinical Validation

Clinical validation of this system was carried out through a series of retrospective and prospective test trials completed in the North and South of Ireland:

site 1. Belfast

site 2. Cork

site 3. Limerick

All sites carried out both ‘Retrospective’ studies using stored samples, and ‘Prospective’ studies using fresh faeces tested in real time.

The specimens were ran in parallel with Reference Lab Taqman® assays as Gold standard (RVL Belfast, NVRL Dublin).

Discrepant results retested with EntericBio and confirmed using gene RealStar®Norovirus RT-PCR Kit 2.0 (Altona) – extraction based internally controlled kit for detection of GI and GII

(Retrospective and Prospective)

Retrospective Trail

Retrospective testing (using stored faecal and vomitus specimens)

Samples which had been positive for Norovirus using routine in-house Norovirus LDT assay were collected and frozen (-80 °C),

Frozen samples were slowly thawed overnight in a fridge and vortexed before testing using EntericBio Realtime Norovirus Assay

Prospective Trail

Inclusion: Routine specimens from patients presenting with symptoms of norovirus infection, and requesting Norovirus studies

Samples were tested using EntericBio Realtime Norovirus Assay in parallel with routine testing using the in-house Taqman ™ assay on the Flow system (Roche) platform on the same day (Belfast), and by Taqman ™ assay at NVRL (Dublin) .

Multicentre clinical validation

Over the three sites a total of 985 samples were tested; 275 retrospectively and 710 prospectively.

Both GI and GII targets amplified, and both faeces and vomit samples were processed during validation.

Discrepant specimens were retested with EntericBio® and also tested alternative CE-marked commercial kit (Altona Real-Star).

Majority of discordant results between both methods had high Ct values.

Non-extraction based

EntericBio PCR:

n=275 PCR Positive PCR Negative

Extraction

based

Reference

Lab

PCR:

PCR Positive 139 4*

PCR Negative 0 132

Retrospective study – frozen samples:

Retrospective results: specificity 1.00; sensitivity 0.97, PPV 1.00 and NPV 0.97

Retrospective discrepant analysis

• 4* samples (1.4%) testing as Negative with EntericBio® were from the one test site.

• All Four were retested through the EntericBio® process and confirmed as negative.

Discrepant PCR analysis with extraction based Altona™ kit - only positive for 1 of the 4, with a high Ct 37.29.

These retest results illustrate that very weak, frozen samples may be borderline in detection, even with extraction based assays.

PCR Analysis

1.Controls

within range

2. Curves smooth

And sigmoidal ‘S’ In shape

3. CT value within accepted range (typically < CT35/36)

Non-extraction based

EntericBio PCR

n=710 PCR Positive PCR Negative

Extraction-

Based

Reference

Lab PCR

PCR Positive 170 3**

PCR Negative 7# 530

Prospective study – fresh samples:

Prospective Results : sensitivity 0.98 , specificity 0.99, PPV 0.96 and NPV 0.99

Prospective discrepant analysis

3 ** samples (0.4%) tested as Negative with EntericBio®

Retested with EntericBio®, one of the three became positive with Norovirus GII.

All three tested positive for Norovirus GII with Altona™ extraction based commercial method.

Of the 7# (0.98%) EntericBio® tested as positive:

four were GI (Ct 30.62, 36.91, 38.13 and 40.63)

No GI repeated with EntericBio®, Ref lab or Altona™.

Total 4 false positive GI

Of the three GII (Ct 20.59, 32.77 and 33.94), only one failed to repeat with either EntericBio® or Altona™(CT 33.94)

Total 1 false positive GII, 2 false negative with LDT

Results Breakdown

Norovirus GI 11% of total Positives

belonged to GI

90% Faeces , 10% Vomit

Ct range –

EntericBio Realtime Norovirus Assay

(17.93-27.43)

In-house Taqman ™ assay (22.19-31.8)

Result Breakdown

Norovirus GII 86% of total positives belonged to

GII

85% Faeces, 15% vomit

Ct range-

EntericBio Realtime Norovirus Assay

(16.33 – 36.68)

In-house Taqman ™ assay (15.12-37.43)

Result breakdown

3 % of total Positive’s had dual infection with both GI and GII NoV

The inhibition rate for EntericBio non-extracted Norovirus PCR was 0.5% (4/710 samples).

Internal control:

EQA Sample EQA Sample

Genogroup Result

LDT Result EntericBio Realtime Norovirus assay ®

GI NoV GII NoV

A GII-4 Positive Ct 21.46 Positive Ct 23.31

B GII-6 Positive Ct 29.39 Positive Ct 23.66

C GII-4 Positive Ct 26.99 Positive Ct 25.97

D Negative Negative Negative Negative

E GI-3 Positive Ct 25.86 Positive Ct 27.69

F GII-4 Positive Ct 26.38 Positive Ct 26.43

G Negative Negative Negative Negative

H GII-4 Positive Ct 26.50 Positive Ct 25.70

I GI-4 Positive Ct 22.94 Positive Ct 31.99

.

Norovirus EQA 2014-2015 (Public Health England)

EntericBio Realtime Norovirus assay ® and in-house

Conclusion of multicenter study

EntericBio Rapid NoV assay less labour intensive

Shorter TAT, no extraction

Rapid detection can lead to better patient management, implementation of appropriate infection control procedures

Can differentiate between NoV genogroups GI and GII, Beneficial for surveillance

CT values available – not black box pos/neg – semi-quantitative

Rapid diagnostics for better patient

management .....

The IPCT should ensure

that faeces specimens from

cases are collected without

delay for norovirus detection,

bacterial culture and, if

appropriate, Clostridium difficile

tests. All microbiological analysis

of stool specimens associated

with potential outbreaks must

be available on a seven days a

week, including holidays, basis.

The turnaround time for

non-culture analysis as measured

from specimen production to

provision of a telephoned or

electronically-transmitted result

should be within the same day

or, at most, 24h in order to

minimize bed closures. Up to a

maximum of six specimens of faeces

from the group of affected

detection in the first instance.

Turn-around-time

0

10

20

30

40

50

60

70

80

90

100

<1 day 1-2 day 2-3 day 3-4 day >4 day

Jan %

Feb %

March %

April %

May %

June %

Transit times Feb 2017

(specimen date to Lims entry/receipt)

0

100

200

300

400

500

600

700

<1 day 1-2 day 2-3 day 3-4 day 4-5 day >5 day

Transit time

Transit time

So how could the RVL gain from EntericBio

Norovirus Assay?

.. Business cases and Tender ...

Northern Ireland: 5 Trusts – Trust Ownership of Noro screening to enable rapid TAT

Outbreak screening

Infection Control

Meet National Guidelines

Support from RVL for elderly care query Rotavirus

RVL to focus on REGIONAL ICU/PICU/NICU, Haematology, Transplant, SCUBU, immunocompromised, immunosuppressed for all 5 targets

Thank you! Christine Meenan,

Microbiology Laboratory,

Royal Victoria Hospital, Belfast

Michael O’Donoghue, Isabelle O’Callaghan,

Liam Blake, Louise Barry, Bartley Cryan.

Microbiology Department,

Cork University Hospital, Cork

Colm McDonnell, Anita O’Connell

Virology Department,

University Hospital Limerick, Limerick

Dave Clancy, Serosep®EntericBio

Monika Lambert, Serosep®EntericBio

Microbiology Belfast

Service BHSCT

Regional service for Virology, TB and Mycology

Microbiology: 680,000 specimens per year (2014) – 410,000 Bacteriology (274,000 Virology-54,000 Molecular, 220,000 serology)

Micro/TB/Mycology: 38 BMS staff, 8 MLA

Virology: 17 BMS (10 Molecular), 5 MLA (2 belong to Micro, on rotation)

Micro/TB/Mycology: 16 Medics: 8 Medical Consultants, 8 Reg grade

Virology: 5 FRCPath Duty Virologists (2 Medical Consultants & 3 Senior Clinical Scientists)

Micro/TB/Mycology: Core Service Hours: 8:00am – 21:00pm, urgents only from 21:00

Virology: Core Service Hours: 8:30am-5:30pm, ‘urgents only’ 5:30pm on

Microbiology Enterics:

EntericBio (March 2015)

EntericBio Gastro panel 2:

Salmonella

Shiga Toxin 1 (Stx 1)

Shiga Toxin 2 (Stx 2)

Shigella

Campylobacter

Cryptosporidium

Giardia

Confirmation:

• Culture

• Microscopy Approx 19,000 faeces/year