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31/05/2017
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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
A12 1 1 2 C+D positive
A13 0 1 3 D negative
A14 1 1 2 D positive
A15 1 1 3 D positive
A16 0 1 3 UV negative
A17 1 1 3 UV positive
A18 1 1 2 D positive
A19 1 1 2 D positive
A20 1 1 3 Co+UV+D positive
A21 1 1 3 D positive
A23 0 1 2 D negative
A25 0 1 3 D negative
A26 1 1 3 D positive
A28 1 1 3 Co+UV positive
A30 1 1 3 D positive
A31 1 1 3 C+D positive
A36 1 1 2 D 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)
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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
A12 2 2 0 0 PR (EURLP) Positive
A16 2 2 0 0 PM (EURLP) Positive
A17 1 2 1 0 PM (EURLP) Negative
A20 2 2 0 0 PR (EURLP) Positive
A28 2 2 0 0 PS Positive
A31 2 2 0 0 PR (EURLP) 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!
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