Sixth Workshop of National Reference Laboratories for Parasites

Istituto Superiore di Sanità, Rome, Italy, 23-24 May, 2011

“Trichinella larvae identification at species level by a molecular method”

1th Proficiency test on

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.

Materials and methodsPOOL OF LARVAE SET

Species N° of vials N° of larvae for each vial

T. spiralis 1 20

T. nativa 1 20

T. britovi 1 20

T. Pseudospiralis 1 20

SINGLE LARVAE SET

Species N° of vials N° of larvae for each vial

T. spiralis 5 1

T. nativa 5 1

T. britovi 5 1

T. pseudospiralis 5 1

No particular restriction regarding molecular technique was established.

Total samples 4

Total samples 20

Lab code Sample type Correct Incorrect N.I. Method used

1 pool of larvae 4 - - EURLP protocol

2 single larvae ? ? ? ?

3 pool of larvae 2 2 - commercial kit based on column + multiplex PCR

4 single larvae 20 - - EURLP protocol

6 pool of larvae 4 - - De Bruyne et al. 2005

8 pool of larvae 3 - 1(TP) Pozio and La Rosa 2003

9 single larvae 13 3 4 EURLP method + German T2/T3 differentiation

10 single larvae 13 - 7 internal method based on EURLP protocol

11 pool of larvae 4 - - commercial kit based on column + multiplex PCR

14 pool of larvae ? ? ? ?

16 pool of larvae 4 - - internal procedure based on Zarlenga et al., 1999

21 pool of larvae 4 - - Pozio and La Rosa 2003

21bis single larvae 11 - 9 Pozio and La Rosa 2003

22 pool of larvae ? ? ? ?

23 single larvae ? ? ? ?

24 pool of larvae 3 1 - EURLP protocol

34 single larvae 4 - 15 commercial kit based on column + multiplex PCR

35 pool of larvae 3 1 - Zarlenga et al., 1999

38 pool of larvae 4 - - Pozio and La Rosa 2003

Results

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 analysisReported 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).

Thanks for your attention