Third Workshop of National Reference Laboratories for Parasites

Istituto Superiore di SanitàRome, Italy

29-30 May, 2008

RING TRIAL

“Detection of adult worms of Echinococcus sp.in intestinal mucosa of the definitive host”

RING TRIAL

Aim of the Ring TrialAim of the Ring Trial

�� One of the tasks of the One of the tasks of the Community Reference Laboratory for ParasitesCommunity Reference Laboratory for Parasites is the is the organization of ring trials for organization of ring trials for NRLs NRLs

�� This year, in addition to a ring trial on This year, in addition to a ring trial on TrichinellaTrichinella, a new ring trial has been , a new ring trial has been organised for the detection of adult worms of organised for the detection of adult worms of EchinococcusEchinococcus sp.sp. in intestinal in intestinal organised for the detection of adult worms of organised for the detection of adult worms of EchinococcusEchinococcus sp.sp. in intestinal in intestinal mucosa of the definitive hostmucosa of the definitive host

�� The aim was to test the skill of the The aim was to test the skill of the NRLsNRLs to detect to detect E. multilocularisE. multilocularis in mucosa in mucosa and to differentiate this worm from other material of parasitic and nonand to differentiate this worm from other material of parasitic and non--parasitic origin present in the matricesparasitic origin present in the matrices

23 NRLs agreed to participate

ParticipantsParticipants

AUSTRIA

BELGIUM

BULGARIA

CYPRUS

ITALY

LITHUANIA

THE NETHERLANDS

POLAND

CZECH REPUBLIC

DENMARK

ESTONIA

FINLAND

FRANCE

GREECE

HUNGARY

IRELAND

PORTUGAL

ROMANIA

SLOVAK REPUBLIC

SLOVENIA

SPAIN

SWEDEN

UNITED KINDOM

Materials and methods Materials and methods -- 11

� The mucosa of the small intestine of 25 foxes found infected with a high worm burden (>100 adult of E. multilocularis) was collected. Ten samples were homogenised to prepare a highly positive sample.

� Foxes shot by hunters in Brandenburg, Germany, in 2007 were collected, stored frozen at -80°C for at least one week and then at -20°until one day before necropsy.

We are very grateful to We are very grateful to Dr. Franz J. ConrathsDr. Franz J. Conraths of Friedrichof Friedrich--Loeffler Institut, Germany, who provided us the Loeffler Institut, Germany, who provided us the 3 samples (codes (codes 11--3) and to 3) and to Dr. Jacek KaramonDr. Jacek Karamon of Department of Parasitology of the National Veterinary Research Institute, Poland, who of Department of Parasitology of the National Veterinary Research Institute, Poland, who provided us the provided us the EchinococcusEchinococcus adult worms useful as adult worms useful as reference materialreference material..

� The same was done with the mucosa with an intermediate worm burden

(25-50 adult parasites) to prepare a weakly positive sample

� Negative mucosa was used to prepare negative sample

Materials and methods Materials and methods -- 22

� 3 samples (negative, weakly positive and highly positive) and the positive control were forwarded to each participant laboratory by an international courier

� The 3 samples were preserved in 0.1% of merthiolate; the positive controlwas preserved in 70% ethanol

� All the samples were delivered within 24-36 hours

�� In each package the following forms were addedIn each package the following forms were added::

Materials and methods Materials and methods -- 33

Form 1Form 1 –– Ring trial result formRing trial result form

Form 2Form 2 -- Protocol for the detection of adult worms Protocol for the detection of adult worms

of of EchinococcusEchinococcus sp.sp. in the intestinal mucosain the intestinal mucosa

Form 3Form 3 –– Laboratory descriptionLaboratory description

Accredited fromAccredited from 1 year1 year 11--5 years5 years > 5 years> 5 years

No. of laboratoriesNo. of laboratories 55 44 55

� 14 laboratories of 23 (61%) are accredited according to ISO/IEC 17025:2005

Results Results -- 11

No. of laboratoriesNo. of laboratories 55 44 55

� 3 laboratories of 23 (13%) have accredited a diagnostic test to detect Echinococcus

larvae/adults in the intermediate/definitive hosts

Results Results -- 22Laboratory Method to detect Echinococcus sp. in definitive host

E1 coproPCRE2 fecal flottation and nestedPCRE4 nrE5 nrE6 IST and PCRE7 ISTE9 coproELISAE10 SCTE11 arecoline surveyE12 nrE13 stereomicroscopeE14 direct examination of dog fecesE15 ISTE16 nrE17 ISTE18 stereomicroscope and PCRE19 nrE20 SCT and PCRE21 stereomicroscopeE22 nrE23 nrE24 SCT or ISTE25 flottation

Results Results -- 33Laboratory Method to detect Echinococcus sp. in definitive host PCR Elisa IST SCT Microscope

E1 coproPCR xE2 fecal flottation and nestedPCR xE6 IST and PCR x xE7 IST xE9 coproELISA xE10 SCT xE11 arecoline survey xE13 stereomicroscope xE14 direct examination of dog feces xE15 IST xE17 IST xE18 stereomicroscope and PCR x xE20 SCT and PCR x xE21 stereomicroscope xE21 stereomicroscope xE24 SCT or IST x xE25 flottation x

0%

5%

10%

15%

20%

25%

30%

35%

40%

PCR Elisa IST SCT Microscope

31,25% 6,25% 31,25% 18,75% 37,50%

Results Results -- 44

Laboratory SAMPLE 1 SAMPLE 2 SAMPLE 3

NEGATIVE HIGLY POSITIVE WEAKLY POSITIVE

E1 POSITIVE NEGATIVE POSITIVE

E2 NEGATIVE POSITIVE POSITIVE

E4 NEGATIVE POSITIVE NEGATIVE

E5 POSITIVE NEGATIVE NEGATIVE

E6 NEGATIVE NEGATIVE NEGATIVE

E7 NEGATIVE POSITIVE NEGATIVE

E9 NEGATIVE NEGATIVE NEGATIVE

E10 NEGATIVE POSITIVE NEGATIVE

3 laboratories (13%) obtained

completely correct results

E11 NEGATIVE POSITIVE NEGATIVE

E12 NEGATIVE POSITIVE POSITIVE

E13 NEGATIVE POSITIVE POSITIVE

E14 NEGATIVE NEGATIVE NEGATIVE

E15 NEGATIVE NEGATIVE NEGATIVE

E16 POSITIVE NEGATIVE NEGATIVE

E17 NEGATIVE NEGATIVE NEGATIVE

E18 POSITIVE NEGATIVE POSITIVE

E19 POSITIVE POSITIVE NEGATIVE

E20 NEGATIVE NEGATIVE POSITIVE

E21 NEGATIVE NEGATIVE NEGATIVE

E22 NEGATIVE POSITIVE NEGATIVE

E23 POSITIVE POSITIVE NEGATIVE

E24 NEGATIVE POSITIVE NEGATIVE

E25 POSITIVE POSITIVE NEGATIVE

Results Results -- 55

Correct

E1 E2 E4 E5 E6 E7 E9 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25

Correct

Uncorrect

Sample 1

Sample 2

Sample 3

Laboratory SAMPLE 1

NEGATIVE

E1 POSITIVE

E2 NEGATIVE

E4 NEGATIVE

E5 POSITIVE

E6 NEGATIVE

E7 NEGATIVE

E9 NEGATIVEE10 NEGATIVE

Results Results –– False positivesFalse positives

7 laboratories (30%) obtained false positive results

Negative correlation between laboratories that use IST E11 NEGATIVE

E12 NEGATIVE

E13 NEGATIVE

E14 NEGATIVE

E15 NEGATIVE

E16 POSITIVE

E17 NEGATIVE

E18 POSITIVE

E19 POSITIVE

E20 NEGATIVE

E21 NEGATIVE

E22 NEGATIVE

E23 POSITIVE

E24 NEGATIVE

E25 POSITIVE

Negative correlation between laboratories that use IST

or SCT methods and the detection of false positive

results (p<0.01)

Results Results –– Why Why false positives?false positives?

Intestinal villus

Results Results –– Why Why false positives?false positives?

Mesocestoides sp.

Images kindly provided by the Polish laboratory

Hookworm

Results Results –– False negativesFalse negatives

Laboratory SAMPLE 2

HIGLY POSITIVE

E1 NEGATIVE

E2 POSITIVE

E4 POSITIVE

E5 NEGATIVE

E6 NEGATIVE

E7 POSITIVE

E9 NEGATIVE

E10 POSITIVE

11 laboratories (48%) obtained false negative results

in higly positive samples

E11 POSITIVE

E12 POSITIVE

E13 POSITIVE

E14 NEGATIVE

E15 NEGATIVE

E16 NEGATIVE

E17 NEGATIVE

E18 NEGATIVE

E19 POSITIVE

E20 NEGATIVE

E21 NEGATIVE

E22 POSITIVE

E23 POSITIVE

E24 POSITIVE

E25 POSITIVE

Results Results –– False negativesFalse negatives

Positive correlation between number of parasites

Laboratory SAMPLE 3

WEAKLY POSITIVE

E1 POSITIVE

E2 POSITIVE

E4 NEGATIVE

E5 NEGATIVE

E6 NEGATIVE

E7 NEGATIVE

E9 NEGATIVE

E10 NEGATIVE

17 laboratories (74%) obtained false negative results

in weakly positive samples

Positive correlation between number of parasites

(highly positive vs weakly positive samples) and

capacity to detect E. multilocularis worms (p<0.01)

E11 NEGATIVE

E12 POSITIVE

E13 POSITIVE

E14 NEGATIVE

E15 NEGATIVE

E16 NEGATIVE

E17 NEGATIVE

E18 POSITIVE

E19 NEGATIVE

E20 POSITIVE

E21 NEGATIVE

E22 NEGATIVE

E23 NEGATIVE

E24 NEGATIVE

E25 NEGATIVE

Results Results –– Why Why false negatives?false negatives?

Images kindly provided by the Polish laboratory

Results Results –– Positive samplesPositive samples

Results Results –– Positive controlPositive control

Final considerationsFinal considerations

The experience derived from this first The experience derived from this first ring trailring trail suggests to use in the future only suggests to use in the future only

frozen material in order to avoid any possible frozen material in order to avoid any possible bias bias while examining samples…..… while examining samples…..…

...but the problem remains the same: the expensive shipping….. ...but the problem remains the same: the expensive shipping…..

The knowledge of the helmintofauna, while examining natural infected animals, is The knowledge of the helmintofauna, while examining natural infected animals, is

important in order to avoid important in order to avoid false positivefalse positive results results

The ring trial evidenced a The ring trial evidenced a positive correlationpositive correlation between between worm burdenworm burden and capacity and capacity

to detectto detect E. multilocularisE. multilocularis

Acknowledgments:

Dr. Franz J. Conraths of Friedrich-Loeffler Institut, GermanyDr. Jacek Karamon of Department of Parasitology of the National Veterinary Research Institute, Poland

Pcr positivePcr positive results in results in negative samplesnegative samples: cross reaction with : cross reaction with MesocestoidesMesocestoides sp.sp.