“Anisakidae worms “Anisakidae worms inducing allergies”inducing allergies”

Mª Teresa Audicana Mª Teresa Audicana BerasateguiBerasategui

Servicio de Alergología e InmunologíaHospital Santiago ApóstolVitoria-Gasteiz (Basque Country, Spain)

EuropeanEuropean UnionUnion ReferenceReference LaboratoryLaboratory forfor Parasites. Parasites.

Roma 2011Roma 2011

AnisakisAnisakis simplexsimplexWho is it?

Where is it?

Anisakidosis

Allergic reactions/diet

Diagnosis

The future

Who is it?

Where is it?

Anisakidosis

Allergic reactions/diet

Diagnosis

The future

Helminths

Plathyhelminthes(flatworms)

Nemathelminthes(roundworms)

Trematoda Cestoda Nematoda

(Intestinal)

Nematoda

(Tissue)

Schistosoma

Fasciolahepatica

Taenia solium

Echinococcusgranulosus

Ascaris

Oxyuris

Anisakis

FilariaTrichinella

spiralis

Anisakidae family: infective genera in humans

• Genus Anisakis– A. typica– A. physeteris– A. simplex– A. brevispiculata– A. shupakovi– A. insignis– A. ziphidarum

• Genus Pseudoterranova– P. decipiens– P. krabbei– P. bulbosa– P. azarasi

• Genus contracaecum– C. osculatum– C. hogmorgini– C. turgidum– C.radiatum– C. mirounga

SNOAPAD

Anisakidosis : familiy

Anisakiosis: Genus Anisakis

• GenusHysterothylacium

•H. aduncum

WAAVPWord Asociation Advancement VeterinaryParasitology (Kassai et al. 1988)

Anisakis simplex larva (L3)

Roundworm 2-3 cm long

Biological cycle

Fish and cephalopods

get infected with the 3rd

larval stage

when they eat

Euphasids

Although they can also

be infected by eating

other contaminated fish

and cephalopods

Audicana and Kennedy (2008) Microbiology Reviews

Who is it?

Where is it?

Anisakidosis

Allergic reactionsdiet

Diagnosis

The future

Abdominal cavity in a parasitized sea fish

Parasites present in edible fishflesh

Fish and fishery productsapparent consumption (FAO 2009)

More than 40 Kg/year per capita Per capita supply(Kg/year)

World 16.8

Portugal 57.2Spain 44.2

Iceland 91China 67Japan 58.6Norway 51,5

European Union 23

AnisakisAnisakis simplexsimplexWho is it?

Where is it?

Anisakidosis

Allergic reactions/diet

Diagnosis

The future

Human infection by Anisakissimplex characteristics

• Humans are accidental hosts

• The majority of cases are due toa single larva

• Usually located in thegastrointestinal tract, although itcan migrate to ectopic locations

• Coprologic studies are notuseful and endoscopy can be diagnostic and therapeutic

Infection Physiopathology

< 1h Adherence tomucosa

Proteolyticenzymesecretion

HaemorrhageTunnelformation

4h - 6d Mucosa andsubmucosapenetration

Chemotacticfactors

EosinophylicphlegmonOedema

7-14 d Granulomaformation

Hypersensitivityresponses

Ulcerouslesions

>14d Larval death Granuloma orpersistentinflammatoryresponse

CureChroniculceration

Anisakidosis has been describedin the 5 continents

• Asia (Korea)• Europe (Holand, France, United Kingdom,

Spain, Germany and Italy among others)• Africa (Egypt)• America (USA including Alaska and Hawaii,

Canada y South American countries) • New Zealand

Anisakidosis is related to fish consumption:rates and cooking habits

Japan

High fish consumption(ussually uncooked:

sushi, sashimi)

58 Kg/person/year

1st case 1965

2000 cases per year(Kagei y cols. 1995).

Spain

High fish consumption(ussually cooked except

“boquerones”)

44 Kg/person/year

1st case 1991

6 cases described (1991-97)

Clinical patterns

1.- Gastric

• 6 to12 h following ingestion

• 72% gastroduodenal

• mainly in major curvature

Anisakidosis:

97% Anisakiosis

3% Pseudoterranovosis

2.- Intestinal

• 48 to 72 h followingingestion

• terminal ilium location

• eosinophylic asciticliquid

3.- Ectopic: mesenteric, pancreatic, hepatic, oropharynx

Anisakiasis: risk fish dishes1. Raw fish dishes:

sushi, sashimi (Japan)gravlax (Nordic)lomi-lomi (Hawaii)ceviche (South America)

2. Smoked at low temperatures, and salted

3. Boquerones en vinagre (Spain)

Raw anchovies preserved in vinegar

Clinical manifestations of anisakidosis

Gastrointestinal:

• acute abdominal pain

• vomiting

• Diarrhoea

Differential diagnosis:

• gastroenteritis

• gastric ulcer

• ileitis

• appendicitis

• acute abdomen

• tumour

Gastroallergic form:Concomitant allergicsymptoms:

• urticaria/angioedema

• anaphylaxis

Diagnosis and treatmentGastric form• Diagnosis

• Anamnesis• Endoscopy• Serologic (IgE)

• Tt: Endoscopy

Intestinal form• Diagnosis:

• Anamnesis• Serologic (IgE)• Radiology• Ascitic punction

• Tt: conservative(non-surgical treatment)

AnisakisAnisakis simplexsimplexWho is it?

Where is it?

Anisakidosis

Allergic reactions/diet

Diagnosis

The future

Food allergy and Rome2000 years ago Tito Lucrecio Caro in “De Rerum Natura”

sayed: “…quod ali cibus est aliis fuat acre venenum”

Food allergy and GI barrier

The GI mucosa is the site of sensitization andchallenge but not necessarily the shock organ

Any organ can be involved but in most cases skinis the first one

Food allergy is mainly a disease of the early yearsof life

The mechanisms of food allergy are closelyrelated to the integrity of the GI barrier

Food as allergens• Low molecular weight proteins (10-70

kDa) and acid isoelectric point (IP)

• Water soluble

• Stability to: high temperature, acid pHand proteolytic enzymes

• High biologic power

• Ussually has a linear structure

Conformational antigens

MastociteMastocite

MastociteMastocite

Degranulation

Sequential antigens

temperature

digestion

acid pH

No degranulation

temperature

digestion

acid gastric secretion

Quantity of allergen

• Sometimes it is possible to induce response by contactand/or inhalation

• The higest phylogenetic distance between allergen andhumans the biggest allergenicity expected (lower doserequired)

Factors involved in food allergyFrom antigen• Enzymatic activity of proteins• Type of food processing (grilled,

cooked, temperature used…)

From patient• Atopic condition• Mucosa permeability• Occupacional exposition: skin,

rhinoconjunctival or bronchial• Risk of severe/fatal anaphylaxis

– Asthma– Drugs (ACE inhibitors, beta blockers)– Mastocytosis

“Compounding factors” EAACI

Concomitant factors that amplify reaction– Exercise– Alcohol, smoke (permeability augmented)– Drugs

• Antacids and inhibitors of protons pompe (lower acid digestion)

• AAS (mucosal dammage)• Beta blockers, ACE inhibitors

– Others: virus, immuno-deficiencies, hormonal changes, other allergensexposition…

Dificult diagnosis

• Hidden allergen

• Cross reactivity

• Panallergens

Anisakis simplex infective larvaeInnate response Adaptative response

NDC

(4) TLRs

T Clonalproliferation

Polyclonal IgE

Mastocytosis

Eosinophilia

Parasite expulsion

MastMast

MDCTh1

Th2

IgG1

IgG4

T

Monoclonal IgE

B

Allergicresponse

Eos

Mast

EosEosEos

TLR1, TLR2,

TLR4, TLR9

ADC

(1)NO

(2)Chemokines

Cytokines

PMN(3)

(5)

(6)

(7)

Mф

TrTh3

Bas

Bas

Ag

Mast

Audicana and Kennedy (2008) Microbiology Reviews

Allergic symptoms of food allergy

Skin:Urticaria/AE

Atopic dermatitis

Systemic:

HipotensionShock

Rhino-conjunctivitis

and/or asthmaDigestivesymptoms

Anaphylaxis is the most seriousallergic reaction I

• Has a rapid onset• The diagnosis is based on defined clinical

criteria (more than one organ involved) • Hypotension and shock are nor necessarily

present• Clinical diagnosis is based on a meticulous

history of an exposure or event• Sometimes but not allways an elevated

tryptase level supported the diagnosis

Anaphylaxis is the most seriousallergic reaction II

Avoidance of the relevant confirmed

allergen trigger is the

key for the optimal management

¿How was our first As allergy case?

• 52 years old woman was referred with four anaphylactic episodes• 3 occurred within 30 minutes after eating hake (Merluccius merluccius). • The last one following preparation of fish for cooking• Tolerated hake and other fish between the episodes• Physical examination: normal. Radiologic and Laboratory tests were

normal • Allergic study negative (foods, drugs, aditives, preservatives...)

• Total IgE: 1.051 kU/l. Specific IgE: Ascaris: 4 kU/l (class 3)• Fecal examination (5 occasions) negative

1st Diagnosis: Idiopathic recurrent anaphylaxis

Hypothesis

¿Is it possible to explain anaphylactic episodes by biological contamination of hake (Merlucciusmerluccius) by Anisakis and to find positivity toAscaris by cross reactivity?

Larvae were collected from muscle tissueof hake (200 mg)

Centrifugation 2 times withsaline solution at 1500 g

Identification

FiltrationLowry proteinquantification 8 mgextract 1/10 W/V 1/100 dilution

prick test

Anisakis simplexthird-stage larvae

Maceration with 2 ml ofsaline solution andcentrifugation

Anisakis simplex extract preparation

Skin tests

Positive specific IgE to Anisakis simplex: 90.8 kU/l (class 5)

Specific IgE (CAP System Phadia)

saline hystamine

A. simplex

Self made extract

0,08 mg/ml

Ascaris: 4 kU/l (class 3)

Diagnosis

2nd: Recurrent anaphylaxis due to Anisakissimplex present as biological contaminant in fish (hake: Merluccius merluccius)Audicana et al. (1995). Recurrent anaphylaxis due to Anisakis simplexparasitizing sea-fish. The Journal of Allergy and Clinical Immunology 96: 558-560.

1st: Idiopathic recurrent anaphylaxis

Alergic reactions to Anisakis• Immediate reaction (first 60 minutes to 4 hours)

• Typical pattern for type I allergic reaction with predominantcutaneous and digestive symptoms

• In 20-60% of cases the symptoms are severe and may affectseveral organs: skin, respiratory, digestive and cardiovascular systems

• Rheumatologic symptoms can appear but are extremely rare

• Airborne and contact antigen in occupational cases– Spain: fishmongers and others (Armentia et al 1998, Audicana 2002)– South Africa: fish-processing workers (Nieuwenhuizen et al 2006)– Italy: fish-degutters (Sanchez et al 2009)

Unexpected characteristics ofAnisakis allergy

• Nonatopic patients

• Average age: over 50 years

Furthermore….

Sometimes elderly people start with ananaphylactic shock (for the first time oftheir lives)

Concomitant use of NAID andsuspicious drug allergy

Name M W (kD) Compartment Function Major allergen Panallergen

Ani s 1 21-24 Excretion-Secretion Kunitz-type trypsininhibitor

YES

YES

YES

Ani s 8 15 Excretion-Secretion SXP/RAL-protein

Ani s 9 14 Excretion-Secretion SXP/RAL-protein

Ani s 10 22 Somatic ? ?

Ani s 11 55 ? ?

YES

Ani s 2 97 Somatic Paramyosin YES

Ani s 3 41 Somatic Tropomyosin YES

Ani s 4 9 Excretion-Secretion Cystein–protease inhibitoras

Ani s 5 15 Excretion-Secretion SXP/RAL-protein

Ani s 6 Excretion-Secretion Serin-protease

Ani s 7 139-154 Excretion-Secretion Glycoprotein

Ani s 12 ? ? ?

Characterized Anisakis simplex allergens

Excretion-secretion (ES) antigens

AniAni s 1 s 1 (24 kD) present in the excretory gland. 86% of thepatients suspicious of an As allergy and parasitationhave positive IgE.Major antigen, potent, sensitive to pepsin and heat

AniAni s 4s 4 (9kD) resistent at heat and pepsin

AniAni s 6 s 6 similar similar sequencesequence toto otherother serinserin--proteinasesproteinasesinhibitorsinhibitors andand inhibitsinhibits chemotripsinchemotripsin

AniAni s 7 s 7 GlicoproteinGlicoprotein reconognizedreconognized by by infectedinfected patientspatients

AniAni s 5 y 8 s 5 y 8 (15 (15 kDkD) ) ThermostableThermostable withwith sequencesequence similar similar toto otherother nematodesnematodes

AniAni s 2s 2 Paramiosin (97 kD) similar to the one present in mites (Blomia tropicalis) Blo t 11

AniAni s 3s 3 Tropomiosin (41 kD) very similar to tropomiosin fromother invertebrates as shrimp (Pen a 1), mites (Der P 10 y Derf 10) and snails. Responsable for skin and CAP tests positive results in asymptomatic cases. Thermostable and available formicroarray.

Somatic antigens

ThermostabilityPrick test: A. simplex extract and heat

1 frozen 2 heated up 3 boiled

Intervalos de confianza del valor de las medias al 95%

Diá

met

ro p

ápul

as

Congelado Calentado Ebullición5,1

5,5

5,9

6,3

6,7

7,11 frozen: extract

2 heated up: 40ºC 20 min

3 boiled: 100ºC 20 min

No significant difference

p= 0.960

Food Drugs Other

Fruit 3 Pain killers 7 Hymenoptera 3

Nuts 2 Contrast media 2 Cholinergic U. 3

Fruit + Nut 4 Pyrazolone 1 Idiopathic 55

Fish 1 Betalactamic 3 A. simplex 8

Shellfish 7 Other 1

Total 17 Total 14 Total 69

PrevalencePrevalence ofof A. A. simplexsimplex allergyallergy in in 100 100 acuteacute urticaria urticaria episodesepisodes

Control group: 100 Blood donors

1

2

• 13% positive in healthyindividuals blood donors

(sensitized)

• Controls (19/150) NOT relevance

13%13%

Del Pozo et al. Allergy 1997

Audicana et al Trends Parasitol 2002

Sensitization: asymptomatic specific IgE

Anaphylaxis

Sensitization: asymptomatic specific IgE

Multicentric Spanish Study: 13% of asymptomatic controlsFernández de Corres et al 2001

Japan: 13% of asymptomatic controlsKasuya et al. 1990

Anaphylaxisreview

Immunoblotstudy

Audicana (2002): PhD ThesisAnaphylaxis 389 cases (Vitoria 1994-1999)

• Drugs

• Foods

• Anisakis

• Hymenoptera venoms

60%

13%

11%

10%

• Nº de episodes/ patient:mean 2 (1-10)

• Emergency room treatment:58 cases (93%)

• Occupationalexposure: 3 cases (5%)– fishmonger– fish road haulage– waiter

0 5 10 15 20 25

20-29

30-39

40-49

50-59

60-69

70-

mujerhombre 61% between

40 and 59 years old

41 ♀21 ♂

mean age is the same in both sexes

Audicana (2002): PhD Thesis61 anaphylactic reactions to Anisakis simplex

Anisakis simplex anaphylaxis: a non-atopic disease

Atopic condition: is not a predisposing factor ≠ common food allergy

casoscontroles

atopia

no atopia

0

20

40

60

80

100

120

140

atopiano atopia

FISH N COOKED UNCOOKED CANNEDHake 23 22 2 -

Anchovies 16 5 11 -

Cod 7 7 - -

Tunna 6 6 - 4

Sardine 3 3 - 1

Skid 3 - - -

horse-mackerel 3 - - -

Megrim 2 - - -

Anaphylaxis: fish species involved

Hake is also the most consumed species in theBasque Country (29,7 g /day)

Prevalenceandintensitydata ofAnisakisparasitismfromdifferentEuropeanareasSource: EFSA 2010

IgE IMMUNOBLOT RESULTS

Immunoblot patterns in cases versus controls (%)

A significant difference in IgE immunoblot with multiplerecognized bands were found in anaphylaxis patiens (a) versus controls (b) (p<0.0001)

23

3

0

10

20

30

1a b

a b

[ ][ ] [ ]

[ ] [ ]CLASE803,0EDAD037,0708,4

CLASE803,0EDAD037,0708,4

e1ecasop ++−

++−

+=

Logistic regression:The better predictor variables of anaphylaxis were:

Age (quantitative value) OR: 1,038 (1,002-1,075)

Specific IgE (class) OR: 2,23 (1,8-2,76)

Calculator

MATHEMATICAL MODEL TO ESTIMATE THE PROBABILITY OF HAVING AN ANAPHYLACTIC EPISODE

Predictor variables

Adult anaphylaxis causes in the 90s N= 625 cases. Audicana 2002 PhD Thesis

389 88

12 62

67

32

5% idiopathic

2% latex

10% parasites60% drugs

13% hymenoptera venom

11% food

10%

37%

15%

25%

13%2%8%

drugs hymenoptera venomfood parasitesidiopathic latex

Adult anaphylaxis causes (2000-2010) N= 669 cases

ECOD. Directive 91/493/EC. September 24, 1991, L 268:15ECOD. Decission 93/140/EC. March 9, 1993, L 56:42 USFDA (1999): Compendium of Fish and Fishery Product Processing Methods, Hazards and Controls. National Seafood HACCP Alliance of Training andEducation. (http.//www-seafood.ucdavis.edu/haccp/compendium/compend.htm)EFSA Journal 2010;8(4) 1543 pp 1-91

How do we kill Anisakis simplex L3 larvaeto prevent infection/anisakidosis?

freezing-15º > 96 hours

-20ºC >24 hours

-35º > 15 hours

Heat treatment>60ºC > 1 min

>63ºC > 15 sec

1. >15 sec 74ºC

2. Cover and turnpieces

3. Repose foodsduring 2 min aftercooking

Microwave

Dietetic guidelines for general population

or

Specific dietetic and safety guidelinesfor allergic patients

Guidelines for general population

Specific for allergic patients:1) Restrictions of seafood:*only for fish and cephalopods*shellfish can be eaten safely (crabs, lobster, prawns, shrimps,

clams, mussels,… etc)2) When eating seafood:* Train the patient to recognize Anisakis in fish in order to remove

it* Eat sea food only at home (avoid restaurants, etc)* Avoid ingesting fish flesh arround the abdominal region* Not to eat fish of small size (example: anchovies)

3) Anaphylaxis: carry IM epinephrine (adrenaline autoinjector) andtraining for use

AnisakisAnisakis simplexsimplexWho is it?

Where is it?

Anisakidosis

Allergic reactions/diet

Diagnosis

The future

Anisakis

T Clonalproliferation

Histamine

MastMast

APCTh1

Th2 T

IgE

B Mast

Immediate reactions (1- 6 h)

TrTh3

Bas

Mast

Skin tests CAP Mast

Bas

CD63BAT

Delayed reactions(> 6 h)

Tryptase

LTT

Diagnostic methodsPatch tests

Challengetests

Audicana and Kennedy (2008) Microbiology Reviews

“In vivo” Skin prick tests

Positive result: wheals > 3 mm with a negative S

The specific IgE Ab on the surface of mast cells, are linked to theantigens and induce the consequent mediator release.

The vasodilatation leads to an increase of permeability and henceresults in oedema

The erythema is due to an axonic reflex

Positive control Histamine (H)

Negative control Saline (S)

Polyclonal conjugated antibodies

Anisakissimplex 1994

IgE

Solid phase: CAP (polymer)

FEIA FluorenzymeImmunoassay(Phadia Diagnostics )

Results: Negative < 0.35 kU/L

Positive > 0.35 kU/L

Class 1: 0.35-1.7

Class 2: 0.7-3.5

Class 3: 3.5-17.5

Class 4: 17.5-50

Class 5: 50-100

Class 6 > 100

“In vitro” Specific IgE determination: CAP FEIA Phadia

0102030405060708090

1-4hours

15 days 1 year 5 years 10 years

IgE (kU/l) Tryptase (ng/ml)

Specific IgE and tryptase levels

BAT: an “ex vivo” test

B CellTH2 Cell

IL4IL13

Live cells from a patient are stimulated by antigens and a biological response is observed

CD40L CD40

TCR MHC

sIgE

StimulantsfMLP, C5a

MediatorsMediators::HistamineHistamine

LeukotrienesLeukotrienes((sLTsLT))

BasophilActivation

CD63 ,others

CytokinesCytokinesChemotacticChemotactic

factorsfactors……

Anisakis

CytokinesCytokines

Anisakis

CD63 expression in a basophil at rest

CD63

Anti IgE FITC orCD123(++) DR (-)

IgE + CD63-

Phenotype IgE positive CD63 negative

Basophil is labelled with Anti IgE FITC or CD 123(++) DR (-)

Ag

IgE + CD63+

Anti IgE FITC orCD123(++) DR(-)

Anti CD63 PEActivation

??

CD63 expression in a activated basophil In activated basophils, the translocation of the proteins in the granules to the membrane is produced and the CD63 is expressed in the membrane

Phenotype IgE positive CD63 positive

TABTAB--AsAs

File: IRENE DEL OLMO ZAPATA.0Sample ID: ANTI IgEGated Events: 660Total Events: 56975

Quad Events % GatedUL 6 0.91UR 556 84.24LL 4 0.61LR 94 14.24

R2R1

Diagnosis by Anisakis Components

Diagnosis by Anisakis Components(only Ani s3 available now)

The microchip is inserted in the reading slide to see theresults by an informatizedanalysis

The results show positives versus negatives and are semiquantitative

There is anallergen in each circle

All allergensare sampledin triplicate

- +

negative positive

AnisakisAnisakis simplexsimplexWho is it?

Where is it?

Anisakidosis

Allergic reactions/diet

Diagnosis

The future

EFSA recomendations (allergy risk)

• Improve surveillance and diagnosticawareness to Anisakis allergic reactions

• Collection of epidemiological data in EU

• Risk asessment of As allergy vs public healthmeasures

• Clear and practical information for clinicians, fishery product handlers and the general public to reduce risks

EFSA recomendations (research)• Mechanisms of allergic sensitization and

exposure

• Differences between geographical regions: exposure and allergy to As

• Infectivity and inactivation of parasites in fishery products

• Studies of safety in fish farming

• Characterization of the risk for fisheryproducts

Other future perspectives

• Stablish dietary and cooking guidelines in general population andallergic patients

• Full molecular characterization of allergens to improve diagnosis by Anisakis Components:– In vivo tests (prick)– In vitro tests (CAP and microarrays)– Ex vivo tests (BAT)

• Certification of allergen free fish products: development of kits to detectallergens in fresh and procesed seafood.

• Occupational allergy. Study of workers risks and protectives measures

• Desensitizacion. Development of vaccines.

“Anisakidae worms inducing allergies”“Anisakidae worms inducing allergies”

Anisakis simplex

• worldwide parasite

• induce severe allergic reactions in adultsand elderly people

• molecular biology cruzial to detectallergens and diagnose patients at risk