“Anisakidae worms “Anisakidae worms inducing allergies” inducing allergies” Mª Teresa Audicana Mª Teresa Audicana Berasategui Berasategui Servicio de Alergología e Inmunología Hospital Santiago Apóstol Vitoria-Gasteiz (Basque Country, Spain) European European Union Union Reference Reference Laboratory Laboratory for for Parasites. Parasites. Roma 2011 Roma 2011
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“Anisakidae worms inducing allergies”old.iss.it/binary/crlp/cont/T_Audicana.pdfOxyuris Anisakis Filaria Trichinella spiralis Anisakidae family: infective genera in humans • Genus
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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
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
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
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