1. Sixth Workshop of National Reference Laboratories for Parasites
Istituto Superiore di Sanità, Rome, Italy, May, 2011
1th Proficiency test on
“Trichinella larvae identification at species level by a molecular method”

2. Introduction
The PT was organized to satisfy the request of some NRLs to be evaluated about their ability to identify Trichinella muscle larvae at species level;
18 NRLS agreed to participate.

3. N° of larvae for each vial N° of larvae for each vial
Materials and methods
POOL OF LARVAE SET
Species
N° of vials
N° of larvae for each vial
T. spiralis 1 20
T. nativa
T. britovi
T. Pseudospiralis
Total samples
SINGLE LARVAE SET
Species
N° of vials
N° of larvae for each vial
T. spiralis 5 1
T. nativa
T. britovi
T. pseudospiralis
Total samples
No particular restriction regarding molecular technique was established.

4. Results Lab code Sample type Correct Incorrect N.I. Method used 1
pool of larvae 4 -
EURLP protocol 2
single larvae ? 3
commercial kit based on column + multiplex PCR 20 6
De Bruyne et al. 2005 8
1(TP)
Pozio and La Rosa 2003 9 13
EURLP method + German T2/T3 differentiation 10 7
internal method based on EURLP protocol 11 14 16
internal procedure based on Zarlenga et al., 1999 21
21bis 22 23 24 34 15 35
Zarlenga et al., 1999 38

5. Results 9 laboratories analyzed pool of larvae;
4 laboratories analyzed single larvae;
1 laboratory analyzed both pool and single larvae;
4 laboratories did not send their results;
Different DNA purification protocols were used;
Almost all laboratories used the multiplex PCR protocol to identify samples, only 1 laboratory amplified and sequenced a portion of the 5S rDNA;
All laboratories that analyzed pool of larvae were able to obtain PCR products;
4 laboratories that analyzed single larvae failed to obtain PCR products from a number of samples between 4 and 15 (20%-75%);
4 laboratories failed the species identification.

6. Single larvae analysis
Reported problems (P), possible causes (C) and suggestions (S)
P: Loss of larva during its transfer from ethanol to water (or PBS) before the analysis.
C: The dried larva tends to float on water surface or to stick to the tip.
S: Transfer the larva directly to the test tube using few volume of ethanol (1-2 µl) and wait the evaporation of the ethanol before starting the DNA purification procedure.
P: Low concentration DNA templates for PCR.
C: The DNA purification method is not enough sensible to be used on single larvae (i.e. kit based on columns with a final elution volume of 200 µl).
S: Use kit based on magnetic beads (more sensible) and decrease the elution volume to µl.
P: Non-specific PCR products.
C: The DNA is not enough purified; unbalanced primers mix; reagents quality.
S: Use commercial kit (better if based on magnetic beads) to purify DNA instead of home made method; check and accurately balance the primers set in the multiplex; use a good Taq polymerase (better if hot start).

7. Conclusions
The mistakes in Trichinella larvae identification observed in this PT were mainly due to the use of not suitable diagnostic tools or protocols and to the lack of appropriate training (mainly concerning single larvae analysis).

8. Thanks for your attention