Diapositiva 1 - Istituto Superiore di Sanitàold.iss.it/binary/crlp/cont/M._Lalle__Anisakis_PTs_2017.pdf31/05/2017 4 Conclusions VI Proficiency Test on the detection of Anisakis spp.

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VI Proficiency Test on the detection of Anisakis spp. L3 larvae in fish fillets

and I Proficiency Test on molecular identification of

Anisakid nematodes at the species level

Marco Lalle

X Workshop of National Reference Laboratories for Parasites 19 May, 2017

Istituto Superiore di Sanità

European Union Reference Laboratory for Parasites Istituto Superiore di Sanità Aim of the PTs

Identification of the presence of Anisakidae L3 larvae in fish fillets

Identification of isolated Anisakids larvae/fragments by molecular

methods (DNA extraction and typing)

The PTs have been organized following the NRL request during the

2016 EURLP Workshop VI

PT on the detection of Anisakis in fish fillets is accredited according to the ISO 17043

The PT on Molecular identification will be submitted for accreditation in fall 2017

PTs time frame

Packages shipped on Monday March 13th

Packages delivery within 72h

PT performed within 3 days from the samples

delivery

Due date to send PT results March 22nd

Due date to send Individual PT report

April 10th

Final PT report will be published on EURLP website after the NRL workshop

PT performed within April 5th

Due date to send PT results April 5th

Due date to send Individual PT report

April 19th

PT detection of Anisakis spp. L3

larvae in fish fillets

PT molecular identification of

Anisakid nematodes at the species level

VI Proficiency Test on the detection of

Anisakis spp. L3 larvae in fish fillets

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Samples and preparation A panel of 3 samples (fish fillet sandwiches) has been delivered to each participant • 2 Fish fillet sandwiches spiked with 1 Anisakidae larva • 1 Fish fillet sandwiches spiked with 3 Anisakidae larvae

Anisakidae L3 larvae were recovered from the body cavity of a heavily parasitized cod from the Mediterranean sea. Fillets of farmed rainbow trout were freshly prepared and used to guarantee an Anisakidae-free matrix.

Detection Methods

A step by step protocol for each method was supplied and any deviation from the main protocol should be reported!

Candling

Compressorium

UV examination after freezing Artificial digestion

The laboratories were allowed to use one (or a combination) of the following methods :

Evaluation criteria

The PT evaluation is only qualitative (presence or absence of larvae). Due to low number of samples and size of the larvae, no statistical analysis of the results is applied. The result is “correct” if the laboratory detected Anisakidae larvae in the three spiked samples The result is “incorrect” if the laboratory did not detect any larva in the spiked samples. The PT is considered “positive” if no “incorrect” results were obtained; the PT is considered “negative” if at least one “incorrect” result was obtained.

Cyprus

Malta

Greece

France (2)

Latvia

Austria

Czech Rep.

Italy

Estonia

Romania

Bulgaria

Belgium

Slovak Rep.

Germany

Finland

UK

Ireland

Sweden

Poland

Lithuania

Spain

Denmark

Hungary

27 Participants: 27 NRLs 26 MS +1 non EU country

PT Participants

Portugal

Slovenia

*first time participant

Iceland*

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C: Candling Co: Compressorium UV: UV ex. after freezing D: Digestion

Results-1

Lab code N° of

spiked/detected larvae1

Method(s)2 Final Evaluation

1 1 3

A1 1 1 2 D positive

A2 0 1 3 D negative

A3 1 1 3 D positive

A4 0 1 3 D negative

A5 1 1 3 D positive

A6 1 1 3 C+D positive

A7 1 1 2 C+UV+D positive

A9 0 1 3 D negative

A10 1 1 3 C positive

A11 0 1 3 D negative



A14 1 1 2 D positive


A16 0 1 3 UV negative

A17 1 1 3 UV positive



A20 1 1 3 Co+UV+D positive





A28 1 1 3 Co+UV positive




Participation 27/27 labs sent the results

Method • 21 Digestion alone or in combilnation with Candeling (3)

• 3 UV alone or in combination with compressorium (1)

• 2 UV +D

• 1 Candeling

labs reported changes in the digestion method:

• 1 “chopped” the fish fillets by stomacher

• 8 chopped” the fish fillets by hands or knives

• 1 performed the digestion at 42°C

• 1 used an internal SOP for which digestion was done in a 2L glass beaker using 0.2 % HCL

and liquid pepsin in a final volume of 1.5L at 35°C for 1h.

• 3 labs, which used artificial digestion, reported on larval viability.

Detection • 8 laboratories failed to pass the PT reporting false negatives

• 9 laboratories underestimate the sample with 3 larvae

Results-2

Corrective Action

The laboratories that failed the PT have been invited to provide, if possible, further details on test execution in order to try to identify the causes of PT failure. The misdetection could be related to: i) package being frozen at the arrival at -45 degrees ii) probable loss of the larva, in the sample spiked with one larva,

due to the improper chopping/manipulation of the sample iii) use of a sieve with mesh size too wide for Anisakidae larva

cause the loss of larvae iv) iv) inexperience of the new analyst v) v) unidentified mistake in the UV-press procedure.

All laboratories agree to participate to new round of test (EQA )

PT Trends (2009-2017)

Type of samples

2009: 1 sample 2013: 3 samples (naturally infected fish) 2014: 3 samples (farmed fish) 2015: 3 samples (farmed fish) 2016: 3 samples (farmed fish) 2017: 3 samples (farmed fish)

Negative Participant at EQA Results not sent

Lab Code 2009 2013 2014 2015 2016 2017

A1 x x x x x x

A2 x x x x x x

A3 x x x x x x

A4 x x x x x x

A5 x x x x x x

A6 x x x x x x

A7 x x x x x x

A8 x x x x x

A9 x x x x

A10 x x x x x x

A11 x x x x x

A12 x x x x x x

A13 x x x x x x

A14 x x x x x

A15 x x x x x x

A16 x x x x x x

A17 x x x x

A18 x x x x x x

A19 x x x x x x

A20 x x x x x x

A21 x x x x x x

A22 x x x x

A23 (A33) x x x x

A24 x

A25 x x x x

A26 x x x x x

A27 x

A28 x x x x

A29 x x x

A30 x x x x

A31 x x x

A32 xA34 xA36 x

AX x

AY x 2009 2013 2014 2015 2016 20160

10

20

30

Participants

Failing Labs

N P

art

icip

an

ts

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Conclusions VI Proficiency Test on the detection of Anisakis spp. L3 larvae in fish fillets

• In comparison with the previous years, the number of participating laboratories decreased (from 20 labs in 2009, 23 in 2013, 27 in 2014, 29 in 2015, 30 in 2016, 27 in 2018).

• No non-NRL labs!

• The performance of laboratory has a general decrease! This is also supported by the underestimation of the number of larvae for the samples spiked with 3 larvae (10/27), irrespective of the method used. As shown during the previous years, inexperience combined with the impossibility to routinely examine fish samples parasitized by Anisakidae generally has a negative effect on the PT performance.

• Low sensitive methods, compressorium and candling, are used only in combination

with artificial digestion or UV method.

• The relative percentage of detection methods adopted did not change substantially over the last years.

• Artificial digestion still remain the method of choice! Largely because it doesn’t require any special equipment.

• UV-press method is applied only in laboratory with a high number of fish samples to be routinely inspected

I Proficiency Test on molecular identification of Anisakid

nematodes at the species level

Samples and preparation

A panel of 4 samples has been delivered to each participant • 2 tubes containing a single fragment of Anisakidae

L3 larva each (A. simplex ss; A. pegreffii) • 2 tubes containing DNA extracted from a single

Anisakidae L3 larva (A. pegreffii; P. decipiens sl) Larvae were collected from body cavity of infected fishes All larvae have been individually identified at species level by analyzing one of their fragments by the EURLP method “Identification at species level of parasites of the family Anisakidae by PCR/RFLP” (http://www.iss.it/binary/crlp/cont/MI_04_website_EN.pdf) The DNAs have been extracted from single larvae and also identified at species level by the above method. Homogeneity is ensured by providing to all participants aliquots of the same DNA preparations.

Detection Methods

“Identification at species level of parasites of the family Anisakidae by PCR/RFLP”

“Identification of Anisakidae Larvae at the species level by multiplex PCR”

Any other suitable molecular method performed by the participant laboratory

(i.e. PCR and sequencing)

rDNA-ITS

ITS

A. pegreffii A. simplex ss

A. simplex/pegreffii Hybrid

A. simplex C

A. ziphidarium

A. physeteris

A. typica

A. sp A

Pseudoterranova sl (P. decipiens s.s.)

Hysterotilacium spp (H. aduncum)

Contracaecum rudolphii (A, B, C)

A. pegreffii A. simplex s.l.

(incl. A. simplex/pegreffii hybrid) A. Physeteris

(incl. A. brevispiculata and A. paggiae)

A. typica

Pseudoterranova sl (P. decipiens s.s.)

Hysterotilacium spp (H. aduncum)

Contracaecum rudolphii (A, B, C)

http://www.iss.it/binary/crlp/cont/MI_04_website_EN.pdf












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Applied method to be describe in the Form 3 (MO/POPVI-00/03.07: Procedure)

List the instruments, reagents and materials used to perform the test to be describe in the Form 2 (MO/POPVI-00/02.07: List of instruments, reagents and materials used to perform the test).

In case you used a published method, indicate the reference and any variation from the original procedure in the dedicated column.

Evaluation criteria

The PT evaluation is only qualitative and no statistical analysis of the results is applied. The result is “correct” if PT items are correctly identified. The result is “incorrect” if PT items are incorrectly identified The PT is considered “positive” if no “incorrect” results were obtained; the PT is considered “negative” if at least one “incorrect” result was obtained.

Greece

France

Italy

Estonia

Romania

Belgium

Finland

UK

Poland

10 Participants: NRLs

PT Participants

Portugal

Results-1

NA, Not applicable. The laboratory didn’t send the results PR=PCR-RFLP PM=PCR Multiplex PS=PCR+Sequencing

Laboratory

code

N° of samples

correctly identified

N° of samples NOT

correctly identified

Method(s) Final evaluation

Larva DNA Larva DNA

A1 NA NA NA NA NA NA

A6 2 2 0 0 PR+PM (Acc. Met) Positive

A7 2 2 0 0 PR+PM (EURLP) Positive

A10 2 2 0 0 PR (EURLP) Positive


A16 2 2 0 0 PM (EURLP) Positive

A17 1 2 1 0 PM (EURLP) Negative


A28 2 2 0 0 PS Positive


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Participation 9/10 labs sent the results

Method • 2 PCR-RFLP and PCR Multiplex

• 4 PCR-RFLP

• 2 PCR Multiplex

• 1 PCR+Sequencing (Cox2 gene)

Method deviation from the suggested • 2 used different DNA extraction kits and gel staining

• 3 reported different Taq polymerases

• 1 used reduced PCR Multiplex (no A. typica or Contracoecum detection)

Detection 1 laboratory failed to pass the PT reporting incorrect identification

Results-2 Corrective Action

The laboratory that failed the PT has been invited to provide, if possible, further details on test execution in order to try to identify the causes of PT failure. The incorrect identification could be related a well to well contamination of the PCR products when the samples were run on an agarose gel.

Conclusions I Proficiency Test on molecular identification of Anisakid nematodes

at the species level

• Participant laboratories proved to be highly competent in the identification of Anisakidae at the species level, irrespective of the molecular method used.

• One laboratory did not send the results due to troubleshoots in ordering material to perform the test.

Thank you for your attention!

Diapositiva 1 - Istituto Superiore di Sanitàold.iss.it/binary/crlp/cont/M._Lalle__Anisakis_PTs_2017.pdf31/05/2017 4 Conclusions VI Proficiency Test on the detection of Anisakis spp.

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