1. Occurrence of Echinococcus multilocularis eggs in environment in endemic region of Poland using molecular techniques
Beata Szostakowska, Anna Lass, Halina Pietkiewicz, Wacław L. Nahorski, Małgorzata Sulima, Katarzyna Kostyra, Sylwia Hallmann, Anna Abramowska, Natalia Cejrowska, Przemysław Myjak
Department of Tropical Parasitology, Institute of Maritime and Tropical Medicine in Gdynia, Medical University of Gdańsk, Poland
Research Project No N N
„The prevalence of Echinococcus multilocularis eggs in environment and attempt to find the correlation between their occurrence and clinical cases of human alveolar echinococcosis”
The aim of work:
Assessment of contamination of environment (soil, fruits & vegatables, water, air) in endemic region of Poland, with eggs of E. multilocularis - to determine the exposure of people to risk of infection
Medical examinations of residents of these areas to establish if the correlation between level of environment contamination and appearance of new (early) human AE cases occurs

2. Epidemiological situation in Poland
Infection of foxes in (Malczewski et al. 2008)
Human AE cases (Nahorski et al. 2013)
Infection of foxes in (Karamon et al. 2014) 9 7 65 11 2 5 3 1
7,9%
1,2%
0,6%
39,6%
6,5%
1,0%
36,8%
2,3%
3,0%
5,6%
4,7%
2,0% 0%
11,8%
28,6%
47,2%
17,5%
5,7%
2,5%
3,9%
30,8%
18,2%
34,0%
50,0%

3. Materials Materials Total number of samples
Around homes (gardens, farmyards)
At a distance form homesteads (forests, fields, etc)
Vegetables/fruits
103
gardens n= 26 , plantations n=21, forests n= 56
Soil 62 27 35
Water 23 12 11
Air 8 3

4. Recovery of eggs (different methods for different materials) & PCR
Soil (400g): drying, sieving (80 g), washing in 0.05% Tween 80, centrifugation
Plants (1/2 kg) : washing, sedimentation, sieving through set of the set of sieves (last: ø50 µm), centrifugation ↓
Conventional flotation using ZnCl2 (specific gravity 1,4), washing out the material from slide to eppendorf tube
Air (1000 l): pumping through gelatinous filter (Sartorius, Germany), transfer of filter to 50 ml Falcon tube, resolving of filter in hot water, washing, centrifugation, transfer of pellet to eppendorf tube
DNA isolation with Sherlock AX kit (A&A Biotechnology, Poland)
Water (up to 50 l): pumping, through capsule filter (Envirochec-HV, PALL corpor.), washing of filter, centrifugation of sediment and transfer to eppendorf tube
DNA isolation with Genomic Mini Kit (A&A Biotechnology, Poland)
Removal of inhibitors with Anty-inhibitor kit (A&A Biotechnology, Poland)
Nested PCR: amplification of fragment of mt 12S rRNA gene

5. Results Material Total number Investigated/positive (%)
Around homes Investigated/positive (%)
At a distance from homesteads
Investigated/positive (%)
Vegetables/
fruits
103/30 (29.1%)
26/9 (34,6%); /4 (19,0%) /17 (30,4%)
Soil
62/7 (11.3%)
27/4 (14.8%)
35/3 (8.6%)
Water
23/1 (4.3%)
12/1 (8.3%)
11/0 (0%)
Air
11/4 (36,4%)
8/3 (37,5%)
3/1 (33,3%)

6. Investigations of inhabitants of endemic areas
Varmia-Masuria province: 272 residents
Pomerania province: residents
410 in total
Ultrasound examination of the abdomen :
Results: cystic lesions of the liver  n = 25
cystic lesions of the spleen n = 1
calcifications of the liver n = 10
calcifications of the spleen n = 2
splenomegaly n = 1
hemangioma of the liver
(in distinguishing  from echinococcosis) n = 2
Serological tests:
ELISA Echinococcus granulosus, ELISA Echinococcus multilocularis, BORDIER AFFINITY PRODUCTS SA, Switzerland
Western Blot ECHINOCOCCUS WB IgG LDBIO Diagnostics, France
Serological tests were negative for all these persons

7. Discussion
Differences in the level of contamination with E. multilocularis DNA of soil, plants, & air are mainly due to different recovery rate, not different level of contamination. Contamination of water is the smallest because eggs are the most dispersed
Results obtained can be underestimated due to imperfection of methods availabe, first of all, recovery of eggs from environmental samples, as well as sensitivity of PCR techniques
Level of contamination of all kind of materials collected around homes was higher → the role of pets in transmission of parasite to humans should be consider
New cases of human AE were not detected. This could be due to the fact that all those who came forward for testing, were examined – maybe not representative population was examined

8. Conclusions Thank you for your attention
The environment of endemic areas should be regularly monitored
Preventive measures should be introduced as education of people especially the most exposed to infection, for example farmers and other village habitants who works at their kitchen gardens, as well as owners of dogs and cats hunting for wild rodents, and people handling foxes
People, who do not live at endemic areas but may spend holidays there should also be aware of the AE threat
Further screening of the population living in endemic areas should be carried out to early detection and treatment of AE
Thank you for your attention