Casulli ring trial [modalit compatibilit ]old.iss.it/binary/crlp/cont/Casulli ring...
Transcript of Casulli ring trial [modalit compatibilit ]old.iss.it/binary/crlp/cont/Casulli ring...
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.