The SSCT method for the diagnosis of Echinococcus multilocularis ...

ANSES Nancy,
National Reference
Laboratory
Franck BOUE
WHO collaborating centre for research on zoonosis
OIE reference laboratory for rabies
CRL for rabies
MINISTERE
DE L’AGRICULTURE
ET DE LA PÊCHE
NRL for rabies and echinococcosis for agriculture minister

SEGMENTAL SEDIMENTATION AND COUNTING
TECHNIQUE (SSCT): AN ADAPTABLE METHOD FOR
QUALITATIVE DIAGNOSIS OF ECHINOCOCCUS
MULTILOCULARIS IN FOX INTESTINES
G. Umhang (1) , N. Woronoff-Rehn (2) , JM. Boucher (1) , V. Hormaz (1) ,
C. Caillot (1) , B. Combes (3) , F. Boué (1) .
(1) ANSES Nancy, National Reference Laboratory, BP 40009, 54220 Malzéville - France
(2) Laboratoire Vétérinaire Départemental du Doubs, 13, rue Gay Lussac- BP 1981- 25020 Besançon Cedex-France.
(3) ERZ, Domaine de Pixerécourt, 54220 Malzéville

QUICK METHOD FOR DEPARTMENTAL VETERINARY LABORATORIES
In the Em national surveillance programme
involving night shooting and analyse of
intestinal content on a large number of foxes
have been examinate.
In this context it has been propose an
optimisation of the initial SCT technique.
Based on previous experience, a modified
SCT method call ‘‘Segmental Sedimentation
and Counting Technique’’ (SSCT), has been
adapted for the routine analysis of a large
number of intestinal content.
The aim was to determine the sensitivity,
specificity, and the parameters of SSCT
method, in order to save time of analysis and
retain the ‘‘gold standard’’ characteristics.

Comparison to gold standard method SCT
SCT
SSCT
S1 S2 S3 S4 S5

Samples distribution
Laboratory Number Qualitative result Worm burden
VDL 227 Yes ND
Anses 131 Yes Yes
Total 358
Neg n =241
67.3%
Pos n =117
32.7%
Classical SCT (Eckert,2003; Hofer ,2000)

Burden charge of Echinococcus multilocularis
50%
40%
The worm burden
distribution has
been determinated
on the 53 positive
intestines analysed
at the French NRL
30%
20%
10%
0%
1 2 3 4 5
S1 S2 S3 S4 S5
Distribution of burden charge of Echinococcus
multilocularis in the foxes intestines
Preferential localisation of Echinococcus multilocularis adult worms
in the posterior part of the intestine in definitive host.

Sensitivity determination for each individual segment
Using the 117 positive intestines, we have determinated the sensitivity of
each segment.
No segment makes it possible to detect 100% of positive intestine

Sensitivity determination for individual and couple of segments
Segment 1 2 3 4 5
1 56,4%
2 72,6% 70,1%
3 83,8% 82,9% 80,3%
4 98,3% 98,3% 97,4% 93,2%
5 73,5% 79,5% 85,5% 94,3% 58,1%
Satisfactory sensitivity of 93.2% in S4 alone
But a high sensitivity of 98.3% is obtained for couples S1-S4 and S2-S4

Segment
Sensitivity determination in function of burden size .
For the 53 positive intestines analysed at the French NRL, the
sensitivity has been determinated in relation with the worm burden.
Sensitivity
Burden charge in the intestine
1-10 11-100 101-1000 >1000
S4 - S1 100,0% 94,4% 100,0% 100,0%
S4 - S2 88,9% 100,0% 100,0% 100,0%
1 intestine failure

Determination of false negative segments
% false negative
Using the 241 negative intestines and negative segments of the positive
animals, we had determinated percentage of false negatives for each segment
20%
18%
17,0%
16,7%
16%
14%
12,7%
12%
10%
9,0%
8%
6%
4%
3,2%
2%
0%
S1 S2 S3 S4 S5
No segment allows a reliability of 100%

False negative determination for individual and couple of segments
Segment 1 2 3 4 5
1 17,5%
2 11,7% 12,7%
3 7,6% 8,0% 9,0%
4 0,8% 0,8% 1,6% 3,2%
5 11,4% 9,0% 6,9% 2,8% 16,7%
Low percentage of false negatives in S4 alone
And a very low percentage of false negatives for couples S1-S4 and S2-S4

S4-S1 and S4-S2 couples characteristics versus Gold std method
Samples find positive
Samples find negative
S4-S1
S4-S2
98,3%
0.8 % of false negative
samples with S4-S1 and S4-S2
With a specificity close to 100 % as the gold standard method.

Time saving
N= 358 Samples
Gold standard: 358 X 5 = 1790 segments analysed.
When we analyze S4 in first and S1 in second we have:
243 Neg 2 X243 =486 segments
109 Pos in S4 109 segments
6 Pos in S1 12 segments
The total with S4 then S1 we analyse in all 607 segments.
Thus 1183 not analyzed segments, we save 66% of time.

Conclusions
SSCT versus SCT gold std method
Time
66% time saving
Sensitivity
Specificity
98.3% 100 %
The SSCT method is a very useful and reliable technique for large
epidemiological studies, particularly in area where the endemic
prevalence of E. multilocularis in definitive hosts is low or unknown.

Use SSCT in a large area where the prevalence is very
low and they have been able to detect new positive animal.

Acknowledgments
I would thank all the partners