Sixth Workshop of National Reference Laboratories for Parasites Istituto Superiore di Sanità, Rome, Italy, 23-24 May, 2011 1th Proficiency test on “Trichinella larvae identification at species level by a molecular method”

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.

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 4 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 20 No particular restriction regarding molecular technique was established.

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

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.

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 50-80 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).

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).

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