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Echinococcus multilocularis infection in animals:

from research project to EU regulation

Adriano Casulli

WHO CC for the epidemiology, detection and control of cystic and alveolar echinococcosis;

European Reference Laboratory for Parasites (EURLP);

ISTITUTO SUPERIORE DI SANITÀ (Rome, Italy)

REGULATION (EU) No 1152/2011:

• Preventive measures for control of E. multilocularis in dogs;

• Ensure continuous protection of Finland, Ireland, Malta and UK who

claim to have remained free of parasite (treatment of pets prior to

travelling);

• Obligation to implement pathogen-specific surveillance programmes

and reports;

• Requirements for the pathogen-specific surveillance programmes.

Legislative Background

The EC has to review 1152/2011 Regulation in the light of scientific

developments regarding Em infection in animals (No later than 5 years

following entry into force) (Dec 2016).

• The European Food Safety Authority (EFSA) has been requested to

provide a scientific opinion on Em infections in animals (Dec 2015).

• To assist in the review, EFSA funded the project “Echinococcus

multilocularis infection in animals” (2012-2015).

Future mandate

E.M.I.A.

consortium

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• Identify and collate the current knowledge and data on 8

Epidemiological points;

• Apply systematic reviews of literature and data.

GP/EFSA/AHAW/2012/01:

Echinococcus multilocularis infection in animals

To prepare for the future opinion (EU 1152/2011):

TIMING: Jan 2013 – June 2015

THE AIM:

Art 36 collaboration project

In order to be able to provide a comprehensive and quantitative

assessment of Em infections in animals, the current knowledge

and data on the epidemiology and risk factors related to this

disease were collected in the EU and ac.

• EMIA consortium

• Board of external experts

Consortium partners

EXTERNAL EXPERTS

CONSORTIUM PARTNERS

COORDINATOR

PC: Adriano Casulli

Thomas Romig,

HU, Germany

Peter Deplazes,

IPZ, Switzerland Bruno Gottstein,

IPB, Switzerland Peter Kern,

UU, Germany

Lucy Robertson,

NVH, Norway

Partner 3:

FLI, Germany Partner 1:

ANSES, France

Partner 2:

RIVM, Netherlands

Partner 4:

EVIRA, Finland

Partner 5:

NVRI, Poland

Partner 6:

CSIC, Spain

SYSTEMATIC REVIEW ADVISORS

SAPIENZA UNIVERSITY, Italy

PROJECT MANAGEMENT ORGANOGRAM

ISTITUTO SUPERIORE DI SANITÀ, Italy

Stage 1 • Project management and a priori Protocols ---------------------------------- Stage 2 • Data collection, evaluation and synthesis ---------------------------------- Stage 3 • Discussions and final drafts of the Project report

WP1

WP6

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EPIDEMIOLOGY (WP2)

• R_2&4: geographic distribution and the prevalence of infection in animals

and the importance of the different hosts species in the life-cycle.

RISK FACTORS (WP3)

•R_1: risk factors and steps for introduction and establishment in free areas;

•R_6: analysis of risk factors associated with human AE;

•R_7: evaluation of impact of infection in animals on public health.

DIAGNOSIS AND TREATMENT (WP4)

• R8: laboratory techniques for the detection in live or dead animals;

• R9: effectiveness of available de-worming drugs and the treatment protocols for pets.

MONITORING, SURVEILLANCE AND CONTROL (WP5)

• R_3: monitoring and surveillance programmes infection in hosts;

• R_5: potential programmes for the eradication in wildlife host species.

TERMS of REFERENCE SR APPROACH

1. a Priori protocols;

2. searching for research studies;

3. selecting studies for inclusion or exclusion;

4. collecting data from the included studies and creating evidence

tabs;

5. assessing methodological quality of included studies;

6. synthesis of data of included studies (meta-analysis);

7. Presenting results.

SYSTEMATIC SEARCH

• Carried out in a centralised way by the Documentation Service of the ISS.

The platform used for this SR is STN International – Fiz Karlsruhe.

• Bibliographic searches, within the 8 a priori protocols on the following databases:

MEDLINE; EMBASE; SCI SEARCH; BIOSIS; CABI; GOOGLE SCHOLAR

• The search was restricted to eight languages from electronic databases (English,

Italian, Polish, Dutch, German, Spanish, French and Finnish).

• Search was carried out on 5/11/2013 and 11/02/2015. The results of the two

searches were combined.

GREY LITERATURE SEARCHING

• EU reports and conference proceedings were searched

• Questionnaires were sent to NRLP in Europe

• Bachelor, Master and PhD thesis searches were carried out using databases:

http://ethos.bl.uk/Home.do;

http://www.dart-europe.eu/basic-search.php;

https://www.daad.de/deutschland/promotion/phd/en/13306-phdgermany-database/;

https://catalogue.lse.ac.uk/Record/1149203;

http://www.theses.fr/;

http://biblioteca.ucm.es/;

http://digital.csic.es/;

https//www.tesisenred.net/;

http://www.proquest.com/en-US/catalogs/databases/detail/abi_inform.shtml;

https://portal.dnb.de/opac.htm;

http://www.sudoc.abes.fr/;

http://www.collectionscanada.gc.ca/thesescanada/;

http://library.stanford.edu/guides/find-dissertations-and-theses;

http://www.oclc.org/support/services/firstsearch/documentation/dbdetails/details/WorldCatDis sertations.en.html;

https://qspace.library.queensu.ca/handle/1974/290.

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Papers identified through databases

searching 10,737

Papers after duplicates removed

5,421

Papers screened

(TITLE AND ABSTRACT) 5,421

Papers excluded (title and

abstract) 4,608

FULL-TEXT PAPERS assessed

for eligibility 813

Duplicates removed 5,316

DATA EXTRACTED from

443 papers

META-ANALYSIS performed

on 362 PAPERS0

PRISMA Flow Diagram (synthesis of 8 SR) EPIDEMIOLOGY (WP2)

• R_2&4: geographic distribution and the prevalence of infection in

animals and the importance of the different hosts species in the life-

cycle.

A spreading and increasing of the disease in animals in Europe is suggested by

recent new findings/expanding of the parasite in several new areas and the

unexpected increase in AE incidence in recognized endemic countries.

There are no studies confirming that dogs and cats can maintain the lifecycle in the

absence of (red) foxes.

KEY FINDINGS (DH):

• 2,805 identified papers, 402 full text, 244 used for meta-analysis.

Studies in 20 countries reported the presence of Em in red foxes, with the

following pooled prevalence (PP):

• Low (≤1%)

(Denmark, Slovenia and Sweden)

• Medium (>1% to <10%)

(Austria, Belgium, Hungary, Italy, Netherlands, Romania and Ukraine)

• High (>10%)

(Czech Republic, Estonia, France, Germany, Latvia, Lithuania, Poland,

Slovakia, Lichtenstein and Switzerland)

• Finland, Ireland, UK and Norway reported the absence of Em in red foxes.

• In Norway (Svalbard), Arctic foxes showed the presence of Em.

Fig. 2 Pooled prevalence of red foxes

at NUTS 1 level (data after 2000).

Fig. 1 Pooled prevalence in red and

Arctic foxes at national level (data

after 2000).

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KEY FINDNGS:

• Raccoon dogs (PP 2.2%), golden jackals (PP 4.7%) and wolves (PP 1.4%) showed

a higher PP than dogs (PP 0.3%) and cats (PP 0.5%).

• For IH, muskrats (PP 4.2%) and arvicolids (PP 6.0%) showed similar PP as

sylvatic DH (excluding foxes).

• Nutrias (PP 1.0%) and murids (PP 1.1%) could play a role in the life cycle of Em

in areas with medium to high PP in red foxes.

• In areas with low PP in foxes, no other DH was found infected with Em.

• For high fox PP, the wolf emerges as a potentially important DH.

• Dogs and cats could be irrelevant in the life cycle of the parasite in Europe.

RISK FACTORS (WP3)

• R_1: risk factors and steps for introduction and establishment in

free areas;

Despite the theoretical ways of introduction of Em into free areas, there are

substantial gaps in the knowledge on the true pathways.

Introduction of the parasite into free areas is necessary but not sufficient for the

establishment of the life cycle.

Appropriate DH and IH must exist to support the life cycle, but the potential role of

environmental factors for the persistence of the life cycle is only partially known.

RISK FACTORS (WP3)

• R_6: analysis of risk factors associated with human AE;

The relative importance of the various PRF for human infection is unknown.

The presumably very long incubation period (10-15 years) for human AE and oral

uptake of tapeworm eggs makes it extremely difficult to study PRF.

Socio-cultural habits from different regions of the world may influence pathways of

infection.

• Observational studies considered: cross-sectional and case-control studies;

• Performed in relation to the different PRF in the included studies;

• The pooled Odds Ratio (OR) were used as a measure of effect;

• The quality of studies was assessed using the Newcastle-Ottawa Scale;

• The Cochrane-Q test to assess the degree of heterogeneity between studies.

METHODS for META-ANALYSIS

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SYNTHESIS of RESULTS

958 identified papers, 73 full text, 29 full text extracted, 17 used for meta-analysis.

17 publications were subjected to meta-analysis:

• CASE-CONTROL studies (n=6) [4 in the EU, 1in China, 1in North America].

• CROSS-SECTIONAL studies (n=11) [10 in China].

POTENTIAL RISK FACTOR N of studies N of participants OR

Dog ownership 5 13,883 2.88 (2.30, 3.62)

Playing with dogs 3 5,916 3.48 (2.20, 5.52)

Hand washing before eating 3 5,348 6.94 (4.99, 9.66)

Gender: female 10 42,812 1.50 (1.35, 1.67)

Age > 20 years 8 24,988 2.96 (2.39, 3.68)

Ethnic group: Tibetan 4 25,952 2.03 (1.56, 2.63)

Low income 2 4,124 3.92 (2.42, 6.36)

Source of drinking water other than well or tap 5 23,714 1.81 (1.52, 2.17)

Occupation: farming 5 17,878 1.29 (0.97, 1.72)

Occupation: herding 5 21,045 2.22 (1.76, 2.81)

Drinking unboiled water 2 7,096 0.63 (0.48, 0.84)

Hunting/handling foxes 3 9,442 1.29 (0.97, 1.71)

Low level of education 2 5,297 4.81 (2.73, 8.48)

CROSS-SECTIONAL STUDIES

with OR and 95 % CI, reporting PRF for human AE (Statistically significant PRF in bold).

CASE-CONTROL STUDIES

with OR and 95 % CI, reporting PRF for human AE (Statistically significant PRF in bold).

Data focusing only on European studies.

POTENTIAL RISK FACTOR

N of studies N of participants OR

Dog ownership 4 1,011 2.30 (1.56, 3.40)

Allowed dog into the house 2 216 1.80 (0.90, 3.62)

Playing with dogs 1 159 2.07 (0.97, 4.42)

Cat ownership 2 265 2.63 (1.42, 4.85)

Living in rural area 2 746 3.12 (1.95, 5.01)

Have a kitchen garden 2 746 5.21 (2.65, 10.22)

Occupation: farming 4 1,011 4.50 (2.74, 7.39)

Did haymaking in meadows not adjacent to water 2 238 3.50 (1.63, 7.55)

Went to forests for vocational reasons 2 266 2.61 (1.13, 6.05)

Ate unwashed strawberries 4 1,006 1.39 (0.87, 2.23)

Chewed grass 2 252 3.20 (1.65, 6.20)

Hunting 4 1,007 1.25 (0.73, 2.15)

Handling foxes 3 902 2.84 (1.57, 5.15)

Eating mushrooms 2 255 0.72 (0.38, 1.39)

Consumption of wild vegetables and fruit 4 990 1.50 (0.98, 2.31)

Protective factors (HLA) 1 604 0.55 (0.34, 0.88)

SYNTHESIS of RESULTS

• The following risk factors of potential global relevance were identified: dog

ownership, playing with dogs, living in endemic areas, female gender, age > 20

years and occupation (herding).

• Other identified potential risk factors were: having a kitchen garden, occupation

(farming), haymaking in meadows not adjacent to water, going to forests for

vocational reasons, chewing grass and handling foxes, whereas particular human

leukocyte antigen (HLA) types turned out to be protective.

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CONCLUSION (and limits)

• The studies were rather heterogenic with regard to the geographic location, study

design, and quality of results…..

• ..…not least because of the long incubation period, which makes the

identification of PRF difficult (drivers? Confounders?);

• It should be emphasized that some of these PRF may represent confounders;

• The results will contribute to draft the review of the current Regulation (EU) No

1152/2011, especially in terms of targeting prevention and control measures

against human AE.

RISK FACTORS (WP3)

• R_7: evaluation of impact of infection in animals on public health;

The true number of cases of AE is not known in Europe mainly because of

underreporting.

Notification of “echinococcosis” is only mandatory in some countries and even

fewer countries have notification requirements on the species level to

differentiate AE from CE.

There is an increase in the number of reported AE cases in new areas, and an

increase of the human AE incidence in recognized endemic countries which

suggests a geographic spread and an increase of the Risk in Europe.

COUNTRY NOTIFICATION of HUMAN AE CASES DIFFERENTIATION BETWEEN CE and AE CASES

Albania -

Austria Yes -

Belgium Yes No

Belorussia Yes -

Bosnia Erzegovina - -

Bulgaria Yes -

Croatia Yes -

Cyprus No -

Czech Republic Yes -

Denmark Yes No

Estonia Yes No

Finland - -

France No No

FYR Macedonia - -

Germany Yes -

Greece - -

Hungary Yes Yes

Iceland Yes -

Ireland Yes -

Italy No -

Latvia - -

Lithuania - -

Liechtenstein - -

Luxemburg - -

Malta Yes No

Kosovo - -

Moldova - -

Montenegro - -

Netherlands No No

Norway Yes No

Poland Yes -

Portugal Yes No

Romania - -

Serbia - -

Slovakia Yes Yes

Slovenia Yes -

Spain Yes No

Sweden - -

Switzerland No -

Turkey - -

Ukraine - -

United Kingdom Yes -

Notification of AE human cases in EU and ac

COUNTRY ESTIMATED ANNUAL NUMBERS of CASES REFERENCES

Albania

Austria 7 Kern et al., 2003; Auer and Aspock, 2001; Torgerson et al., 2010

Belgium 1 Torgerson et al., 2010

Belorussia 6 Torgerson et al., 2010

Bosnia Erzegovina

Bulgaria 1 Torgerson et al., 2010

Croatia

Cyprus

Czech Republic 1 Torgerson et al., 2010

Denmark

Estonia 9 Torgerson et al., 2010

Finland

France 21 Abdullaev et al., 2006; Torgerson et al., 2010

FYR Macedonia 1 Torgerson et al., 2010

Germany 61 Torgerson et al., 2010

Greece 1 Torgerson et al., 2010

Hungary 1 Torgerson et al., 2010

Iceland

Ireland

Italy

Latvia 9 Torgerson et al., 2010

Lithuania 9 Torgerson et al., 2010

Liechtenstein

Luxemburg

Malta

Kosovo

Moldova 1 Torgerson et al., 2010

Montenegro

Netherlands

Norway

Poland 3 Torgerson et al., 2010

Portugal

Romania

Russia 1.180 Torgerson et al., 2010

Serbia

Slovakia 4 Torgerson et al., 2010

Slovenia 2 Torgerson et al., 2010

Spain

Sweden

Switzerland 20 Torgerson et al., 2010

Turkey >100 Torgerson et al., 2010

Ukraine 10 Bessanov et al., 2003; Torgerson et al., 2010

United Kingdom

Estimation of annual number of AE human cases in the EU and ac

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DIAGNOSIS AND TREATMENT (WP4)

• R8: laboratory techniques for the detection in live or dead animals;

Two main approaches are used for the diagnosis of Em in foxes: the SCT and

DNA-based methodologies.

Studies on the efficiency of the diagnostic tests for detection of Em in live or dead

animals are very heterogenic, which complicates drawing any conclusions from

them.

It has been demonstrated that there is a gap in evaluating diagnostic test with

scarce information on the sensitivity of tests, even those considered as the main

important to detect the parasite in definitive host populations.

Lack of standardization of diagnostic methods detecting Em probably causes

variation in sensitivity and specificity between labs.

DIAGNOSIS AND TREATMENT (WP4)

• R9: effectiveness of available de-worming drugs and the treatment

protocols for pets.

Due to its favorable pharmacokinetic properties and activity against both immature

and mature stages, praziquantel is the substance of choice for the treatment of

Em.

No information was found about the time of egg/parasite dispersal after treatment,

so no recommendation can be given how long after treatment feces need to be

discarded.

MONITORING, SURVEILLANCE AND CONTROL (WP5)

• R_3: monitoring and surveillance programmes infection in hosts;

There is no requirement for the monitoring or surveillance of Em in non-free

countries in the EU.

Risk-based surveillance is permitted but difficult to implement due to the limited

understanding of the risk factors.

There is considerable spatial and temporal heterogeneity in Em distribution within a

country and across Europe. Therefore, the results of local or regional surveys

cannot be extrapolated to a whole country.

MONITORING, SURVEILLANCE AND CONTROL (WP5)

• R_5: potential programmes for the eradication in wildlife host

species.

Eradication of EM in the European wildlife has not been achieved in areas where

foxes are present.

Long term control but not elimination of the parasite may be possible by baiting.

Increased fox hunting/trapping is not considered to be effective in controlling the

parasite.

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• More oriented to EU regulation and data modelling…..

EFSA: Animal Health and Walfare panel Em status of EU MS and adjacent countries

Uncertain endemicity: freedom not documented but no case reported

MS Test used Test sensitivity as reviewed by

Reported method

(Sensitivity)

Cited Ref

by MS

Casulli et al.,

(2015)

Conraths et al.,

(2015)

FINLAND

MC-PCR (fishing Real time PCR

targetting 12S rRNA gene

(78% by internal validation)

Isaksson et al.,

2014 88% - 95.7%

88%

(compared to Isaksson et

al., 2014 on RT-PCR)

IRELAND SCT (Se 98%) Eckert, 2003 98% 83.8%

(Eckert et al., 2001)

MALTA

Sieving/flotation of faecal samples for

copro-egg detection and Multiplex-PCR

targetting 12S rRNA and nad1 genes

(Se 94%)

Mathis et al.,

1996 88% - 95.7%

50%

(Trachsel et al., 2007)

UK

sieving/flotation of faecal samples for

copro-egg detection and modified PCR

Cest1-Cest2 targetting nad1 mithocondrial

gene (proposed Se 85%)

Mathis et al.

1996;

Dinkel et al.,

1998

88% - 95.7% 89%

(compared with IST)

NORWAY

MC-PCR (fishing Real time PCR

targetting co1mithocondrial gene)

(Se 63%)

Øines et al.,

2014 88% - 95.7%

88%

(Isaksson et al., 2014)

Uncertainty around the diagnostic sensitivity of the different tests used

by the labs involved in the surveillance programmes under Regulation

(EU) No 1152/2011.

GRAPHICAL REPRESENTATION used to estimate the probability

of introduction from an endemic area of origin to free areas.

NWILDi is N of foxes moving from the sub-area to a free area

ρWILDi is the true prevalence in the sub-area

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CONCEPTUAL MODEL:

probability of introduction,

transmission and establishment

of Em in a free country.

0 200 400 600 800 1000

0.0

0.2

0.4

0.6

0.8

1.0

Probability of Introduction

Wild canids

Number of foxes crossing the border

Pin

tro

WIL

D

0.4

0.30.1

0.050.010.001

Prevalence in

country of origin

PROBABILITY OF INTRODUCTION (P_introWILD) as a function of the number

of wild canids moving from an endemic area to a free area and of the hypothetical

true prevalence of infected foxes in the adjacent endemic area where the animals

come from (different colours)

TRELLIS PLOT with 16 DIFFERENT SCENARIOS

X-axis=number of dogs/foxes. Y-axis=probability of introduction.

Red line: dogs & border checks in place.

Blue line: dogs & no border checks in place.

Green line: foxes.

MAIN CONCLUSIONS and RECOMMENDATIONS

• Red fox as principal DH in Europe;

• No evidence that any other carnivore can maintain the lifecycle of Em;

• Rodents and other IH are not suitable target species for surveillance;

• Movements of infected DH is an important introduction pathway;

• Lack of standardization in diagnostic methods (diagnostic sensitivity needed!);

• Until better documentation is available, the diagnostic sensitivity should be set

conservatively at 78%.

• ENCOURAGE

• Studies targeting: PRF (enabling risk-based sampling), environmental influence on

Em life cycle;

• Notification of human cases and differentiation at species level (case definition!).

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http://www.efsa.europa.eu/en/supporting/pub/882e

Experts working group: Adriano Casulli, Franz Conraths, Helen Roberts, Helene Wahlström, Rene Bødker and Thomas Romig

http://www.efsa.europa.eu/en/efsajournal/pub/4373

Regulation (EU) No 1152/2011

by EC

SCIENTIFIC REPORT

by Grant Consortium (E.M.I.A.)

EXPERT OPINION

by EFSA, AHAW panel

New EU regulation!

by EC

• The research that led to these results has received funding from the EFSA (Echinococcus

multilocularis infection in animals) g.a. GP/EFSA/AHAW/2012/01.

Thank you!