utility of viral pathogen testing as a regulatory parameter for eu … · 2013. 9. 24.  ·...

Utility of viral pathogen

testing as a regulatory

parameter for EU bathing

waters

Peter Wynn-Jones, Jane Sellwood, Mark Wyer and David Kay

Policy

Context

Projects

Virobathe (FP6)

Epibathe (FP6)

Viroclime (FP7)

Activites 1. Methods development (Virobathe)

Filters vs glass-wool and bovril vs marvel

2. Inter-laboratory AQC

3. Correlations with health-related FIOs

4. Acqusition of surveillance data

1. Fresh

2. Marine

3. Sewage

1. Raw

2. Secondary

3. tertiary

5. Assessment as a regulatory parameter

Viroclime

Adenovirus results grouped by quartiles of Escherichia coli

concentration - marine water

p = significance of Mantel-Haenszel linear trend

test not significant at the 95% confidence level (p > 0.05)

* the colour shading

indicates the proportion

of results in each

category

Adenovirus results grouped by quartiles of Intestinal

enterococci concentration - fresh water

p = significance of Mantel-Haenszel linear trend

† test significant at the 95% confidence level (p < 0.05)

* the colour shading

indicates the proportion

of results in each

category

Categorical analysis of Adenovirus results - Summary, quartile

interval, conservative

p = significance of Mantel-Haenszel linear trend

† test significant at the 95% confidence level (p < 0.05)

Parameter Type p

Escherichia coli Fresh †0.000

Escherichia coli Marine 0.164

Intestinal enterococci Fresh †0.000

Intestinal enterococci Marine 0.793

Somatic coliphage Fresh †0.000

Somatic coliphage Marine 0.461

Norovirus GGII Analysis

928 Fresh water analyses (232 “tetrads”)

482 Marine water analyses (121 “tetrads”)

Conservative - PCR fail = 0

Affects 5.0% of fresh water results

and 7.9% of marine results

Categorical analysis of Norovirus GGII results

n

Fresh - 232

Marine - 121

* the colour shading

indicates the proportion

of results in each

category

Italic figures at the base of the bars =

number of positive “tetrads”

Norovirus GGII results grouped by quartiles of Intestinal

enterococci concentration - marine water

p = significance of Mantel-Haenszel linear trend

test not significant at the 95% confidence level (p > 0.05)

* the colour shading

indicates the proportion

of results in each

category

Categorical analysis of Norovirus GGII results - Summary,

quartile interval, conservative

p = significance of Mantel-Haenszel linear trend

† test significant at the 95% confidence level (p < 0.05)

Parameter Type p

Escherichia coli Fresh †0.002

Escherichia coli Marine 0.050

Intestinal enterococci Fresh 0.054

Intestinal enterococci Marine 0.361

Somatic coliphage Fresh †0.000

Somatic coliphage Marine 0.106

Ring Trials Can two labs give comparable answers on the same

sample?

Results of Mineral Water Matrix Ring Trial

May 2011

UMU and FIOCRUZ

have mean values

which are not

statistically different

Results of Sewage Matrix Ring Trial

May 2011

All labs have mean

values which are

statistically different

Advice Lack of correlation with known FIOs

Testing by qPCR was not reproducible with the

precision needed for regulatory parameter

Wide swings (peaks) in key pathogens such as

Norovirus

Indicates pathogens shedders in the population NOT

‘risk’ in a precautionary assessment

Needs more epidemiology

Swansea Bay Model

Validation

November 2015

Mark Wyer, David Kay and the SCSC Team

Swansea Bay model NRW Hydrometry

inputs

SCSC Installation

Inputs

Calculated Variable Inputs

Black Pill – UVA,

wind speed

Clyne River -

discharge

Afan - discharge

Dulais - discharge

Workbook

Output

Sign:

Good

Poor

Without discounting = SUFFICIENT

Discounting using the model/signage:

2013 – 2 samples discounted/0 replaced

2014 – 2 samples discounted/2 replaced

2015 – 1 sample discounted/1 replaced

Official classification = GOOD

A clear demonstration of the utility of applying

prediction and discounting under Directive 2006/7/EC

2012-2015 Classification

DSP Intensive sampling

Pronounced

variation within

the sampling day

Water quality - pGI Thresholds

Water quality thresholds relating to pGI can be

developed using relationships between Mean log10

enterococci and pGI

Asymptotic sigmoid

functions –

Richard’s Curve –

fitted to data

Site specific model

for Swansea DSP

2014 validation data

20 days

1 day in each week of the bathing season

15/05/2014 – 30/09/2014

30-minute sampling between 08:00 and 17:00 BST

19 samples per day

Sampled as per 2011

Enterococci analysis using triplicate filtrations to

improve precision – as per 2011

Results – Individual samples

Familiar pattern of variation in IE concentration within each

sampling day

Results – Daily GM

Variation in daily geometric mean IE concentration:

the measure predicted by the model

Comparison of daily results - GM

Comparison with 2011 results – statistically

similar

Comparison of daily results - SD

Comparison with 2011 results – No statistically

significant change

Diurnality – AM vs PM

GM IE concentration was lower in the afternoon

Difference was statistically significant overall

and when daily pGI > 0.1

2014 model fit

Model over predicts in late September?

Within-season decline in solar radiation – near equinox?

Problems with UVA sensor shading?

2014 model fit

8 points above

the line (P=0.4)

12 points below

the line (P=0.6)

Binomial test for

Ho: P=0.5

p=0.5034

Model fit: 2011 & 2014 data

2011:

29 points above

the line (P=0.47)

30 points below

the line (P=0.51)

Binomial test for

Ho: P=0.5

p=1.0000

Performance - Misclassification

2011

2014

Critical misclassification:

≈ 5%

Thanks

City and County of Swansea Pollution Control Division

staff

Natural Resources Wales Analytical Services staff

Patrick Lilly – Welsh European Funding Office

Strathclyde Loch Microbial

Tracer Study

November 2015

Mark Wyer, David Kay and the CREH team

Background

Strathclyde Loch was the venue for the prestigious triathlon events in the Glasgow 2014 Commonwealth Games

An illness outbreak, attributed to norovirus, was recorded following a competitive event in summer 2012 - reported by John Logan at the Southport Symposium

A curtain barrier system was deployed to partition a swim area for the events

A microbial tracer experiment was designed to test the effectiveness of the barrier against virus particles

Barrier curtain system

Tracer organism

Large plaque phage isolated from sea water

Infects Serratia marcescens bacteria

Plaques – clear areas

where the phage has

infected the host bacteria

Units: plaque forming

units (pfu)/ml

Study design

Prior sampling showed absence of the tracer organism

in the loch

Continuous hourly sampling at 9 locations around the

loch for 72 hours – 11th – 14th November 2013

1 litre of tracer released where South Calder Water

enters the loch

1.0 x 1016 particles of S. marcescens bacteriophage

released

Nine sampling sites

Sampling

Measures taken to avoid potential cross contamination

by the sampling team itself

A system of completely independent sampling

implemented for the sites in the two areas

Two sets of dedicated sampling equipment – clearly

marked and cleaned after each sample

Samples from inside and outside the swim zone were

stored and transported in separate cool boxes

Outside swim zone - south

Outside barrier at pontoons

Tracer “breakthrough” 23 h after release

Inside swim zone - south

Inside barrier near causeway to tower

Tracer present in one sample (2 pfu/ml) 31 h post release

Suggests the curtain barrier is very effective at this site

Swim zone – south east

Inside barrier adjacent to the erstwhile overflow to the Clyde

Tracer present in two samples (1 pfu/ml) 44 and 63 h post release

- Suggests the curtain barrier is very effective here

Outside swim zone - north

Outside barrier near rowing course finish marker

Tracer “breakthrough” 18 h post release

Inside swim zone - north

Inside barrier at rowing course finish marker

Tracer “breakthrough” 25 h post release – leakage past barrier

Indicates that the barrier not as effective at this site

Reasons

Incomplete sealing of the barrier at the shore – physical

connection observed at the start of the study

Blow over from waves generated in the larger area of

the loch was observed during sampling

Closure of the two sides of the barrier observed during

the study

Suggests the barrier requires management at this side

of the loch to be fully effective

Tracer transfer by loch waterfowl?

Thanks

Ian Spiers – Scottish Government

Calum McPhail - SEPA

Brian Devlin – North Lanarkshire Council

Sandyhills Bay Tracer and

Water Quality Study

November 2015

Mark Wyer, David Kay and the CREH team

Study Design

Release of 4 microbial tracer organisms to inputs discharging to Sandyhills Bay – bacteriophage

5 litres of tracer at each site

MS2 coliphage – 5 x 1017 particles

Serratia marcescens phage – 4.3 x 1016 particles

fX174 bacteriophage – 1.45 x 1017 particles

Enterobacter cloacae phage – 6 x 1015 particles

Continuous hourly sampling at at the beach for 54 hours: 22nd – 24th September 2013

Analysis for faecal indicator organisms, turbidity and salinity

Sampling

Wide tide range and rapid tidal movement required use

of an ATV on the beach to cover all states

Tracer release and sampling

Additional sediment sampling

Results – MS2 Barnhourie Burn

Tracer appears 4-5 h after release

Present in most subsequent samples

Elevation on alternating tides?

Results – S. marcescens phage Sandyhills Car Park Septic Tank

Tracer appears 5 h after release

Present in most subsequent samples

Strong signal on the next incoming, remaining through low water

Results – fX174 phage

Powbrade Burn

Tracer appears 9 h after release

Present in all subsequent samples

Pattern appears to relate to tide – higher concentration at HW

Results – Ent. cloacae phage Southwick Water

Tracer appears 12 h after release

Present in all subsequent samples

Initial peak at next HW, gradually decaying on subsequent tides

Results – phage tracers in sediment

MS2 and S. marcescens phage from Barnhourie Burn present in

samples from both low tides

Higher MS2 reflects higher tracer titre

Results – phage tracers in sediment

Ent. cloacae phage from Southwick Water not present in samples

from from the first tide – consistent with the tracer time series

fX174 from Powbrade Burn present on both tides

Results – FIOs Day vs. Night

No statistically significant differences (Student’s t-test p > 0.05)

Results – FIOs inside/outside bay

Statistically significant differences (Student’s t-test p < 0.05)

GM FIO concentrations were higher inside the bay

Results – FIOs in sediment

FIOs were present in sediments – higher levels of enterococci

(104cfu/100g) cf. E. coli (5x103 cfu/100g)

Difference in survival characteristics?

Results – Turbidity & salinity

Clear pattern of reduced salinity associated with the incoming

tide and through HW – freshwater trapped in the smaller bay by

the incoming tide?

Results – inside/outside bay

Mean salinity significant different inside the bay

(Student’s t-test p < 0.05)

No significant difference in GM turbidity

Conclusions

Tracers demonstrate connectivity between all 4 labelled

sources and the DSP through successive tidal cycles

Tracer patterns appear to reflect the tidal sequence

FIO concentrations show tidal rather than diurnal (i.e.

day night) relationships

Samples collected within the bay had significantly

higher FIO concentrations and significantly lower

salinity than those collected outside

Conclusions

The pattern suggests that water with a relatively high

freshwater component and FIO concentration from the

adjacent catchment is trapped in the smaller bay area

on incoming tides

Microbiological analysis of sediments showed presence

of FIOs

Tracer organisms – were also deposited in the

sediments – probably through adsorption to sediment

particles

Thanks

Ian Spiers – Scottish Government

Calum McPhail – SEPA

Ruth Stidson – SEPA

David Findlay – SEPA

Andy Thomas – Gillespie Leisure

Further information

Professor David Kay:

dave@crehkay.demon.co.uk

Dr Mark Wyer:

mark@crehwyer.demon.co.uk