Alveolar Echinococcosis of the Liver with a Rare Infiltration of the Adrenal Gland.

Human alveolar echinococcosis (AE) is a silently-progressing disorder that has become a threat in many countries. Since 2000, when the first case was recorded, the number of human AE patients in Slovakia is on continuous raise. The article presents a rare case of alveolar echinococcosis with infiltration in the adrenal gland and discusses the problems associated with differential diagnosis of the disease. In 2016, abdominal ultrasound performed due abdominal pain complaint showed the presence of cystic lesions in the right liver lobe of 54-year old female patient. During surgery, another lesion in the right adrenal gland was found, and neoplastic processes or echinococcosis were considered in the differential diagnosis. Due to unclear correlation between radiology, serology and histopathology results and endemic situation in Slovakia, molecular examination was recommended. Subsequently E. multilocularis was confirmed as etiological agent of infection. Alveolar echinococcosis is considered as a rare disease, with very few patients referred to clinicians or hospitals that sometimes have almost none existing experience with the diagnosis and treatment of the disease. Therefore, the establishment of networks or reference centres specialized on management of the disease would be suitable way to provide the patients with the best care and improve the disease diagnosis, treatment and prognosis.

A Comparative Study of Different Immunoassays to Detect Specific Antibodies to Echinococcus Spp. in Human Sera.

Human echinococcosis, one of the most serious of parasitic zoonoses, is caused by the larval stages of taeniid cestodes of the genus Echinococcus. The study aimed to assess the reliability of the detection of specific antibodies to E. multilocularis and E. granulosus s.l. in human sera and to compare their diagnostic potential for their utilization in the practice. In the study, the somatic antigen of E. multilocularis (AgEm), antigen B (AgB), and the hydatid fluid antigen of E. granulosus and two commercial ELISA kits - Echinococcus granulosus (Bordier Affinity Products, Crissier, Switzerland) and NovaLisaTM Echinococcus IgG (NovaTec Immunodiagnostica, Germany) - were compared. Sera of patients with alveolar and cystic echinococcosis, and with different parasitic/other infections were used to evaluate the sensitivity, specificity and cross-reactivity of in-house and commercial ELISA methods. AgEm presented the highest values regarding the diagnostic indicators, showing 100 % specificity and 90.0 % sensitivity. The tests for serological diagnostics of cystic echinococcosis were less sensitive and specific. The Echinococcus granulosus kit had 83.8 % specificity and 88.2 % sensitivity, while AgB and AgHF showed 85.0 % and 86.3 % specificity, and 76.5 % and 100 % sensitivity, respectively. NovaLisaTM Echinococcus IgG proved to have 95.7 % specificity and 77.8 % sensitivity. The results point out that the combination of different serological tests and approaches in accordance with clinical and imaging findings is still essential to prove the correct diagnosis in suspected patients.

The fox tapeworm, Echinococcus multilocularis, in grey wolves and dogs in Slovakia: epidemiology and genetic analysis.

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is an important emerging parasite in the northern hemisphere. In epidemiological studies, the highest attention is being paid to foxes as the main reservoir hosts responsible for geographic expansion from multiple focal populations and the invasion of urban habitats, but little information is available on the parasite distribution in other carnivores. Hence, the study was designed to obtain updated information about the occurrence and genetic diversity of E. multilocularis in grey wolves and dogs in Slovakia. Faecal samples of wolves were collected from three locations under a certain level of environmental protection in the central and eastern parts of the country, and the presence of the parasite DNA was detected in 35.7% of 112 samples, with the highest rate (51.2%) recorded in the Poloniny National Park in north-eastern Slovakia. Among 110 faecal dog samples, E. multilocularis was detected in three faeces from segregated Roma settlements in the eastern part of the country, which accounted for an overall positivity of 2.7%. Sequence analysis of two mitochondrial genes, 12S rRNA and NADH dehydrogenase subunit 1, revealed four haplotypes in 13 isolates from wolves and dogs originating from four sites in eastern and central Slovakia, with all samples bearing a European-type pattern of E. multilocularis. The more than one-third positivity rate of E. multilocularis in wolf faecal samples dispersed over a large part of the country has corroborated the extensive circulation of the parasite in wildlife and confirmed the need to improve intervention control strategies.

The Mouse Bile Duct Tapeworm, Hymenolepis Microstoma in Free-living Small Mammals in Slovakia: Occurrence and Genetic Analysis.

The mouse bile duct tapeworm Hymenolepis microstoma, is a potentially zoonotic species with a wide variety of reported definitive hosts of rodent genera. In the present study the occurrence of H. microstoma in free-living small mammals in selected areas of Slovakia and the retrospective analysis of epidemiological data published in Slovakia were performed. Hymenolepis microstoma was detected in two animal species, the common shrew (Sorex araneus) and the European hamster (Cricetus cricetus) of 186 small mammals examined from two ecosystems, urban and natural ecosystem of national park. No mention about the presence of this parasite in Slovakia in the past was found following a bibliographical search. Partial sequences of the nuclear paramyosin gene showed the shrew isolate placed in a subclade together with H. microstoma from Portugal, with high bootstrap value for its differentiation from the sister species Hymenolepis nana. Similarly, the analysis of the nuclear ribosomal ITS region placed the hamster isolate in the cluster composed of H. microstoma from Australia, Spain and Portugal. The Slovak isolate was the most distinctive sample among available H. microstoma, differing in 1.4 - 1.9% of nucleotides from the remaining isolates. The difference (seven of 17 nucleotide positions) was partially due to indel polymorphisms associated with two and five nucleotides. To our knowledge, these are the first reports of H. microstoma in Central Europe and also the first record of infection in the common shrew. A recently indicated zoonotic potential of H. microstoma along with a possibility of its direct transmission between animals and/or humans without the need of intermediate hosts pose a public health concern in contaminated areas of Slovakia. The use of molecular techniques may substantially facilitate more thorough understanding of the epidemiological situation of H. microstoma and related tapeworms in various ecosystems of the country.

Global Distribution of Alveolar and Cystic Echinococcosis.

Alveolar echinococcosis (AE) and cystic echinococcosis (CE) are severe helminthic zoonoses. Echinococcus multilocularis (causative agent of AE) is widely distributed in the northern hemisphere where it is typically maintained in a wild animal cycle including canids as definitive hosts and rodents as intermediate hosts. The species Echinococcus granulosus, Echinococcus ortleppi, Echinococcus canadensis and Echinococcus intermedius are the causative agents of CE with a worldwide distribution and a highly variable human disease burden in the different endemic areas depending upon human behavioural risk factors, the diversity and ecology of animal host assemblages and the genetic diversity within Echinococcus species which differ in their zoonotic potential and pathogenicity. Both AE and CE are regarded as neglected zoonoses, with a higher overall burden of disease for CE due to its global distribution and high regional prevalence, but a higher pathogenicity and case fatality rate for AE, especially in Asia. Over the past two decades, numerous studies have addressed the epidemiology and distribution of these Echinococcus species worldwide, resulting in better-defined boundaries of the endemic areas. This chapter presents the global distribution of Echinococcus species and human AE and CE in maps and summarizes the global data on host assemblages, transmission, prevalence in animal definitive hosts, incidence in people and molecular epidemiology.

Seroprevalence of Trichinella infections in domestic dogs from Slovakia.

Trichinellosis is constantly present in the sylvatic cycle in Slovakia, with several sporadic human outbreaks registered since the 1960s. The largest outbreak of trichinellosis occurred in 1998 and was related to the consumption of dog meat that had been added to smoked pork sausages. The aim of this study was to estimate the seroprevalence of Trichinella infection in domestic dogs in various regions of Slovakia. Out of 439 dogs tested, 56 (12.8%) were classified as a seropositive based on the enzyme-linked immunosorbent assay (ELISA), with confirmation by Western blotting. The highest seropositivity was recorded among dogs from the eastern part of Slovakia, in the Presov (22.9%) and Kosice (17.1%) regions, long considered to be highly endemic for Trichinella occurrence and where the prevalence of infection in the vulpine population is also the highest in Slovakia. Trichinella-seropositive animals were detected significantly more often in dogs kept in rural areas (21.1%) when compared with animals living in towns or suburban localities (7.7%). On the other hand, neither age, nor gender and size of the dogs were shown to be a significant factor for Trichinella infection (P > 0.05). The present results indicate that the dog population in Slovakia is at high risk of exposure to Trichinella parasites and could potentially represent a suitable host group for serological monitoring of Trichinella infection as convenient sentinel animals.

Seroprevalence of human Toxocara infections in the Roma and non-Roma populations of Eastern Slovakia: a cross-sectional study.

Socioeconomic conditions and health of the Roma population, the most numerous minority in Europe, are worse than that of the non-Roma population. Information about the occurrence of human toxocarosis and other parasitic diseases in the Roma population is scarce or completely missing. The aim of this study was to map the seroprevalence of toxocarosis in the population living in segregated Roma settlements and to compare the data with the occurrence of antibodies in the non-Roma population of Eastern Slovakia. The seropositivity to Toxocara in 429 examined Roma inhabitants of segregated settlements reached 22·1%, while only 4/394 samples of the non-Roma population were found to be positive (odds ratio 27·7, P < 0·0001). Headache, muscle pain, influenza-like symptoms and diarrhoea occurred significantly more often in seropositive persons than in seronegative individuals. In the Roma population positivity was not influenced by gender, level of education and poverty, but age, lack of sanitary facilities and heating with wood significantly increased the risk of infection. It can be assumed that besides the high prevalence of toxocarosis, other parasitic diseases and communicable diseases will also be more prevalent in the Roma population living in segregated settlements.

Alveolar echinococcosis in a highly endemic area of Northern Slovakia between 2000 and 2013.

Long-term surveillance of Echinococcus multilocularis occurrence in red foxes in Slovakia revealed the existence of highly endemic areas, with an overall prevalence rate of 41.6 % in the northern part of the country. Between 2000 and 2013, 26 human cases of alveolar echinococcosis were detected and only three of them were not in endemic localities in northern Slovakia. Remarkable is the occurrence of the disease in eight people younger than 35 years, including three patients aged eight, 14 and 19 years. Occurrence of E. multilocularis in red foxes throughout the country and high incidence of alveolar echinococcosis in young people indicate high infectious pressure in the environment of northern Slovakia. It can be assumed that the real incidence of alveolar echinococcosis is significantly higher than recorded by official data due to the lack of existing registration and reporting system. For effective management of prevention and control strategies for this disease improvement of the national surveillance system and engagement of specialists outside the medical community are necessary. Our study presents a comprehensive picture of the epidemiological situation of E. multilocularis in northern Slovakia. In addition, we report the first list of confirmed human cases of this serious parasitosis in Slovakia.

Small mammals: paratenic hosts for species of Toxocara in eastern Slovakia.

Toxocara spp., an aetiological agent of a serious helminthozoonosis, is a common roundworm of domestic and wild carnivores worldwide. The study aimed to estimate the seroprevalence of Toxocara in small mammals from different localities in eastern Slovakia. Anti-Toxocara antibodies were detected in 6.4% out of 2140 examined animals trapped in eastern Slovakia. Due to their high density and observed high seroprevalence of toxocariasis, Apodemus agrarius, A. flavicollis, Myodes glareolus and Mus spicilegus (10.9, 4.2, 3.6 and 11.2%, respectively) represent important sources of the infection. A significant correlation between type of food and Toxocara positivity was detected: granivores (7.2%) and invertebratophages (7.1%) were positive more frequently than herbivores (2.1%). In the years monitored, cyclic changes of seroprevalence were observed. A higher prevalence of antibodies in the spring was followed by a decrease in summer. In autumn, seroprevalence started to rise and stayed at a similar level through the winter. Seroprevalence of the examined animals confirms their contact with Toxocara spp. and demonstrates the presence of the aetiological agent in the monitored locality. Areas with a high prevalence of infected animals present constant infectious pressure on definitive hosts, thus also increasing infection risk for humans and paratenic hosts. The study confirmed the contact of small mammals with Toxocara spp. and demonstrated the presence and circulation of an aetiological agent in the localities monitored in eastern Slovakia.

Influence of Echinococcus multilocularis infection on reproduction and cellular immune response of mice.

The influence of secondary Echinococcus multilocularis infection on reproduction and cellular immune response of mice was studied in BALB/c mice infected with 2000 E. multilocularis protoscoleces. Of the total infected mothers, 11.7% did not give birth and 10% of uninfected ones did not deliver. Both, healthy and infected mothers, produced on average 6-7 offspring per litter. The changes in production of seral IFN-gamma, TNF and IL-10 did not significantly influence the course of gravidity. On the other hand, more intensive metacestode growth was observed after the delivery. This study confirmed the ability of host organism to adapt to severe damage caused by E. multilocularis, not only in normal conditions, but also during pregnancy.

Dirofilaria infections in working dogs in Slovakia.

A monitoring programme aimed at the diagnosis of subcutaneous dirofilariasis and heartworm disease in working (police and military) dogs in Slovakia has been performed during the period of September 2007 to February 2008. In co-operation with the Ministry of the Interior and the Ministry of Defence, in total, 710 dogs (591 police dogs and 119 military dogs) were investigated for the presence of microfilariae in blood. All police and military dogs in active service held on the territory of Slovakia were included. Microfilariae were detected in 118 (20.0%) police dogs and 10 (8.4%) military dogs. The most infected individuals originated from southern parts of Slovakia (Trnava region 53.6% and Nitra region 39.6%); the prevalence was low in northern regions (Zilina 3.1% and Presov 6.6%). In several districts of southern Slovakia, the prevalence of subcutaneous dirofilariasis in working dogs exceeded 40%. In all infected animals, the autochthonous origin of the disease was confirmed; however, due to the frequent movement of working dogs, it was not possible to identify the exact locality of infection. At present, a dog living in Nemsová village in Trencín district (north-western part of the country) is regarded as the northernmost localized autochthonous case of subcutaneous dirofilariasis in Slovakia. In three dogs, co-infection of Dirofilaria repens and Dirofilaria immitis was detected. High prevalence rates in working dogs and the zoonotic characteristic of the disease represent an undoubtedly important veterinary and medical problem that requires the urgent introduction of prophylactic and control measures.

Neospora caninum, potential cause of abortions in dairy cows: the current serological follow-up in Slovakia.

Neosporosis is considered to be a contributing risk factor for abortions in dairy cows and other farm animals and has negative economic impact on their breeding. In respect of the rapid spread of neosporosis in herds throughout the world, our aim was to detect the prevalence of anti-Neospora antibodies in cows post-abortion (PA) (PA Group n=716) and in cows without any reproduction problems (Control Group n=247) on large breeding farms from Eastern Slovakia. The overall mean seropositivity in PA Group (20.1%) was significantly higher (p<0.0001) in comparison with the Control Group (2.3%), and this demonstrates the causal dependency of abortions on neosporosis. On farms from south-east region, 15.6% of cows were seropositive on average ranging from 7.8% to 25.8% in different districts. In the northern region, significantly (p=0.0002) higher 26.2% mean seropositivity was detected. The seroprevalence in different districts ranged from 2.9% to 39.4%. In the PA Group a high seropositivity to infectious bovine rhinotracheitis (41.5%) and sporadic occurrence of Q-fever, leptospirosis and chlamydiosis was also detected. From 11 Toxoplasma-seropositive cows, only one animal was simultaneously positive to Neospora. The relatively high prevalence rate in cows warrants the attention and a need of surveillance in Slovak herds. A better knowledge of epidemiology of this etiological agent and the mechanisms of its transmission may help in the introduction of more effective preventive and control measures.

The first finding of Echinococcus multilocularis in dogs in Slovakia: an emerging risk for spreading of infection.

Alveolar echinococcosis, caused by the metacestode Echinococcus multilocularis, is a zoonosis of public health significance. Apart from red foxes, other carnivore species can also serve as definitive hosts of this parasite. This study aimed to determine the occurrence of E. multilocularis in dogs in the Slovak Republic and identify risk factors for acquiring the infection. Out of 289 dogs examined, E. multilocularis was detected in eight samples (2.8%). Positive results were observed in shepherd dogs (12.5%), guard dogs (7.1%), hunting dogs (2.4%) and in one dog with unknown usage (2.5%). Catching rodents and feeding with raw viscera were confirmed to be the most important risk factors for E. multilocularis infection. On the contrary, the way of dog use, frequency of going to the rural areas, the age and gender of animals seem not to be risk factors for infection. No significant association was observed between the positivity of dogs coming from high endemic or non-endemic regions. This is the first report of E. multilocularis in dogs in the Slovak Republic that confirms substantial contribution of dogs to the transmission of the tapeworm in this territory and urges the necessity of prophylactic measures for dog owners.

Humoral immune response of mice infected with low doses of Trichinella spiralis muscle larvae.

Serological techniques are frequently used to detect parasite status and to monitor epidemiology and disease prevalence in important reservoir hosts of zoonotic diseases. Small mammals present the most important link in the epidemiological chain in the spread of trichinellosis. In experimental studies, high infective doses are used to provoke strong immune response of laboratory animals. Wild animals, however, could be infected with very low numbers of Trichinella larvae. The aim of this work was to reveal the size of infective doses that can evoke an adequate immune response with detectable level of specific antibodies in mice. Sixty inbred (Balb/c) mice were infected with 50 L1 and 60 outbred (ICR) mice were infected with 5 L1 T. spiralis. The total larval burdens (TLB) in the intestinal and muscle phases, reproductive capacity index (RCI), and the kinetics of development of specific antibodies by iELISA with different conjugates were determined. In the first 10 days post infection (dpi), more adults were found in the intestines of inbred mice. In both mice strains, the first muscle larvae were observed at 20 dpi. The RCI was significantly higher in outbred mice. Sero-conversion of IgM antibodies was detected at 30 dpi. The IgG antibodies appeared at 40 dpi in inbred mice, and at 50 dpi in outbred mice. Using a polyvalent conjugate, the earliest sero-conversion was recorded at 30 dpi. Antibody levels increased until the end of the experiment (80 dpi). Our results support the suitability of ELISA in large epidemiological surveys to detect low-level infection in naturally infected small mammals, and are useful in epidemiological studies of the sylvatic circulation of trichinellosis to determine likely modes of transmission.

Field evaluation of an intravital diagnostic test of Echinococcus multilocularis infection in red foxes.

Echinococcus multilocularis parasitizes the small intestine of red foxes (Vulpes vulpes) and other carnivores, and has a wide distribution throughout the northern hemisphere. This cestode is the causative agent of human alveolar echinococcosis, a life-threatening helminth zoonosis. In 2000-2002, 2130 red foxes were examined for its presence in Slovakia, with a total prevalence of 30.7%. The data on occurrence were obtained by the combination of necropsy of small intestines from red foxes and coproantigen detection in faecal samples. The correlation between the number of detected specimens and the value of optical density of copro-ELISA test was found. When worm burdens were low (1-25 specimens) the sensitivity of the method was 31.3+/-8.64%, when worm burdens were >50 specimens, 81.8+/-0.66%, and with high worm burdens (>1000 specimens) the sensitivity reached 100+/-0.34%. E. multilocularis presence was detected using the nested PCR method from the eggs in the faecal samples with a 100% specificity. In epidemiological surveys of this zoonosis, it is of crucial importance to detect animals with a high level of infection, which are responsible for the bulk of environmental contamination. The advantage of copro-ELISA test lies in allowing the intravital diagnostics to be employed within the epidemiological survey of E. multilocularis occurrence in the protected and urban areas.

Circulation of Toxocara spp. in suburban and rural ecosystems in the Slovak Republic.

Toxocara spp., the common roundworms of domestic and wild animals, are the causative agents of larval toxocarosis in humans. The migration of Toxocara larvae in men causes clinical syndrome, called larva migrans visceralis or larva migrans ocularis. The objective of the present work was to investigate the prevalence of toxocarosis in dogs, red foxes (Vulpes vulpes), and small mammals in the Slovak Republic. T. canis infection was diagnosed in 16.6% out of 145 dogs examined. Young animals showed significantly higher positivity (50.0%) than adults (12.4%). Coprological investigation of 310 red foxes showed 8.1% prevalence of T. canis, with higher occurrence in animals from rural conditions (8.6%) than from suburban environment (5.6%). Out of 710 small mammals examined, anti-Toxocara antibodies were detected in 7.7% of the animals. The most frequently seropositive species was Apodemus agrarius (15.9%). Seropositivity of small mammals from suburban localities was higher (10%) compared with rural areas (5.8%), with adult animals exhibiting higher seropositivity (8.0%) than subadults (6.8%). Our results have confirmed the importance of dogs, red foxes and small mammals in circulation of this serious helminthozoonosis in suburban and rural ecosystems.