Cystic echinococcosis: Development of an intermediate host rabbit model for using in vaccination studies.

The aims of this study were an establishment of the domestic rabbit as an intermediate host for cystic echinococcosis (CE) and to evaluate the potency of the crude germinal layer and the protoscoleces antigens to protect against the CE. Firstly; Two groups of white Newzeland rabbits were infected orally either by 5000 active oncospheres or viable protoscoleces separately. After 20 weeks, the slaughtered rabbits showed the presence of hydatid cysts at different internal organs. Molecular detection of the resulted cysts was conducted. Secondly; 27 rabbits were divided into nine groups (n = 3). Groups 1 and 2 were immunized with the crude germinal layer antigen while the groups 3 and 4 were immunized with the crude protoscoleces antigen. Groups 5 and 6 received the adjuvant mineral oil. Groups 7 and 8 were used as positive control. The last 9 group was kept as a negative control. The obtained results showed a significant high protection percentage of 83.4% and high antibody titer was recorded in groups that received the crude germinal layer antigen comparing with the groups that immunized with the crude protoscoleces antigen as their protection percentage was 66.7% with lower IgG response. In conclusion, the domestic rabbits could be used as a laboratory model for CE. Developing of the germinal layer antigen is more immunogenic than the protoscoleces one and could be used as a promising vaccine. Attention should be directed towards the existing rabbit in the environment adjacent to infected dogs as it could be a part of Echinococcus life cycle.

Multi-Epitope Fusion Protein Eg mefAg-1 as a Serodiagnostic Candidate for Cystic Echinococcosis in Sheep.

Cystic echinococcosis (CE) in sheep is a hazardous zoonotic parasitic disease that is caused by Echinococcus granulosus (Eg). At present, serological test is an important diagnostic method for Eg infection in domestic animals. Here, a fusion protein Eg mefAg-1 harboring 8 dominant B-cell epitopes of Eg such as antigen B, tetraspanin 1, tetraspanin 6, reticulon and Eg95 was produced in E. coli and evaluated for CE in sheep by indirect ELISA. Eg mefAg-1 showed in ELISA a high sensitivity (93.41%) and specificity (99.31%), with a coincidence rate of 97.02%. Overall, it is suggested that the Eg mefAg-1 could be a potential antigen candidate for CE serodiagnosis in sheep.

Granulomatous responses in larval taeniid infections.

Granulomas are responses to persistent nonliving bodies or pathogens, centrally featuring specialized macrophage forms called epithelioid and multinucleated giant cells. The larval stages of the cestode parasites of the Taeniidae family (Taenia, Echinococcus) develop for years in fixed tissue sites in mammals. In consequence, they are targets of granulomatous responses. The information on tissue responses to larval taeniids is fragmented among host and parasite species and scattered over many decades. We attempt to draw an integrated picture of these responses in solid tissues. The intensity of inflammation around live parasites spans a spectrum from minimal to high, parasite vitality correlating with low inflammation. The low end of the inflammatory spectrum features collagen capsules proximal to the parasites and moderate distal infiltration. The middle of the spectrum is dominated by classical granulomatous responses, whereas the high end features massive eosinophil invasions. Across the range of parasite species, much observational evidence suggests that eosinophils are highly effective at killing larval taeniids in solid tissues, before and during chronic granulomatous responses. The evidence available also suggests that these parasites are adapted to inhibit host granulomatous responses, in part through the exacerbation of host regulatory mechanisms including regulatory T cells and TGF-ß.

Hydatidosis: Preparation and evaluation of radiolabeled antigens and antibodies.

The present preliminary study intends to evaluate the in vitro use of hydatid antigen and their antibodies once labeled with iodine 125(I125) and characterized from viewpoint of radiochemical purity and immunoreactivity. Radiolabelled molecules gave satisfactory purity of 94% and 96%-98%, for hydatid antigen and IgG respectively. As regards, the specific activity of these latter, varied between 4.79 and 5.97 µCi/µg. The specificity test of radiolabelled IgG against the hydatid membranes showed a significant recognition that increased proportionally according to the contact surface. Likewise this immunoreactivity test performed with a simple binding assay, using human hydatid fluid antigen (HHF-Ag), previously fixed on a solid phase, gave satisfactory fixation rate of the order of 356 ±â€¯48.08cpm, 2539 ±â€¯550.12cpm and 6558 ±â€¯712.76cpm for the concentrations of 0.1 µg/ml, 2 µg/ml and 25 µg/ml respectively. Statistical study of 88 sera, carried out with radiolabelled antigen (125I-HHF-Ag) in competitive radioimmunoassay test (CRIA) showed highly significant difference (p < 0.0001) in the binding capacity of antigens from patients sera with hydatid disease (65.63 ±â€¯9.12) compared to the negative sera (19.25 ±â€¯14.84). No cross reaction was observed using sera from patients with toxoplasmosis (33, 07 ±â€¯13, 07) and the difference was highly significant (p < 0.0001) compared to E granulosus infected patient sera. Furthermore, this test seemed to be sensitive since among the 43 sera tested, only 37 (86%) were found to be positive by passive hemagglutination (HAP), while the totality (100%) responded positively by CRIA. Our findings are encouraging, suggesting that these radiolabeled molecules could be useful for advancing toward new diagnostic and therapeutic modalities.

Immunization of rhesus macaques with Echinococcus multilocularis recombinant 14-3-3 antigen leads to specific antibody response.

E. multilocularis (Em) is the etiologic agent of alveolar echinococcosis (AE), a severe and potentially fatal disease, primarily affecting the liver of and occurring in aberrant intermediate hosts, e.g., humans and non-human primates. Due to increasing numbers of spontaneous cases of AE in the Old World monkey colonies of the German Primate Center, the question arose as to whether vaccination of non-human primates may represent a useful prophylactic approach. In this pilot study, the recombinant antigen Em14-3-3, which has provided a 97 % protection against E. multilocularis challenge infection in rodent models, was used for the first time to immunize rhesus macaques. In order to increase immunogenicity, the antigen was formulated with different adjuvants including Quil A®, aluminum hydroxide (alum), and muramyl dipeptide (MDP). Also, different vaccination regimens were tested. All vaccinated animals developed antigen-specific antibodies. While Quil A® induced a local adverse reaction, alum proved to be the most potent adjuvant in terms of induced antibody levels, longevity as well as tolerability. In conclusion, our pilot study demonstrated that recombinant Em14-3-3 is safe and immunogenic in rhesus monkeys. As a next step, efficacy of the vaccination remains to be explored.

Regulation of immunity by Taeniids: lessons from animal models and in vitro studies.

Taeniidae is the largest family of the Cyclophyllidea order of parasites despite being composed of just two genera: Taenia spp and Echinococcus spp. These parasites are flatworms with a terrestrial life cycle, having an immature or larval stage called metacestode, which develops into the mature form within the intestine of the primary host after being consumed in raw or poorly cooked meat. Consumed eggs hatch into oncospheres, penetrate the intestinal walls and are transported via the bloodstream to later develop into metacestodes within the muscles and internal organs of secondary and sometimes primary hosts, thereby initiating the cycle again. Larval stages of both Taenia spp and Echinococcus spp are well known to produce tissue-dwelling, long-lasting infections; in this stage, these parasites can reach centimetres (macroparasites) and both genera may cause life-threatening diseases in humans. Establishing such long-term infections requires an exceptional ability to modulate host immunity for long periods of time. In this review, we analyse the immunoregulatory mechanisms induced by these tapeworms and their products, mainly discussing the importance of taeniid strategies to successfully colonize their hosts, such as antigen-presenting cell phenotype manipulation and the consequent induction of T-cell anergy, among others.

Correlation of serum sHLA-G levels with cyst stage in patients with cystic echinococcosis: is it an immune evasion strategy?

Patients with cystic echinococcosis (CE) can harbour cysts for years or even decades, apparently without effect of the immune system on the metacestode. Although several immune evasion mechanisms by echinococcal cysts have been described, it is unclear whether the human leucocyte antigen (HLA) system plays a role in the susceptibility or resistance to CE in humans. HLA-G molecules are known to exert a suppressive action on dendritic cells maturation and on natural killer (NK) cells functions, therefore hampering T-cell responses and NK cytolysis. HLA-G plays an important role in immune tolerance, is involved in foetus and in allotransplant tolerance, and may be involved in tumoral and viral immune evasion. In this study, we assessed the presence and levels of soluble HLA-G (sHLA-G) in patients with CE using a commercial ELISA kit to determine whether host's HLA-G may have a role in the course of human CE.

Diagnostic parameters of serological ELISA for invasive amoebiasis, using antigens preserved without enzymatic inhibitors.

Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemagglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis.

Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

The laminated layer: Recent advances and insights into Echinococcus biology and evolution.

The laminated layer is the unique mucin-based extracellular matrix that protects Echinococcus larvae, and thus to an important extent, shapes host-parasite relationships in the larval echinococcoses. In 2011, we published twin reviews summarizing what was known about this structure. Since then, important advances have been made. Complete genomes and some RNAseq data are now available for E. multilocularis and E. granulosus, leading to the inference that the E. multilocularis LL is probably formed by a single type of mucin backbone, while a second apomucin subfamily additionally contributes to the E. granulosus LL. Previously suspected differences between E. granulosus and E. multilocularis in mucin glycan size have been confirmed and pinned down to the virtual absence of Galß1-3 chains in E. multilocularis. The LL carbohydrates from both species have been found to interact selectively with the Kupffer cell receptor expressed in rodent liver macrophages, highlighting the ancestral adaptations to rodents as intermediate hosts and to the liver as infection site. Finally, LL particles have been shown to possess carbohydrate-independent mechanisms profoundly conditioning non-liver-specific dendritic cells and macrophages. These advances are discussed in an integrated way, and in the context of the newly determined phylogeny of Echinococcus and its taenid relatives.

Anaphylactic shock ensuing therapeutic puncture of an echinococcal cyst.

Cystic echinococcosis (CE) is a widespread zoonosis. For treating single echinococcal cysts during the last decades, therapeutic puncture of the cyst, aspiration, injection of a scolicide, and re-aspiration (PAIR) has been established as a minimal-invasive alternative method to surgery. A recent review on the complications of therapeutic cyst punctures has shown that dangerous complications occur much less frequently than previously assumed. A case is described where an allergic acute bronchospasm and arterial hypotension led to a life-threatening shock immediately after echinococcal cyst puncture. Fortunately, the situation could be managed by an experienced and well-equipped anesthesiology team. Life-threatening allergic phenomena after puncture of echinococcal cysts may occur less frequently than generally assumed; nevertheless, they must be taken into account, and precautions must be taken to manage serious adverse events.

Prevalence of Serum IgG Antibodies to Cystic Echinococcus Antigen among Patients in an Uzbekistan Emergency Hospital.

Cystic echinococcosis (CE) is one of the most widespread zoonotic helminthiases, which can last an asymptomatic infection for several years. The purpose of this study was to demonstrate serum antibody prevalence of CE among asymptomatic people in Uzbekistan using ELISA. A total of 2,547 serum samples were collected, 66 from confirmed CE patients and 2,481 of patients with other diseases than CE at a hospital in Tashkent, Uzbekistan. The serum samples were screened for CE specific IgG antibodies by ELISA using cystic fluid antigen obtained from sheep. The serum antibody positive rate was 89.4% (59/66) in CE and 3.6% (89/2,481) in other disease patients. The present ELISA recognized 89.4% sensitivity and 96.4% specificity. The ELISA absorbance of positive samples was distributed 0.271-0.971 for CE and 0.273-0.887 for other disease patients. The other disease patients with high absorbance over 0.3 were 50 (2.0%) who were presumed to be active CE patients. The patients in their 40s showed the highest positive rate of 5.2% (P=0.181), and women were 4.4% while men were 3.1% positive (P=0.136). The data confirmed that there are many asymptomatic patients of CE in Tashkent. It is indicated that CE is an endemic disease of public health importance in Uzbekistan.

Detection of hydatid-specific antibodies in the serum and urine for the diagnosis of cystic echinococcosis in patients from the Kashmir Valley, India.

Serological diagnosis of cystic echinococcosis (CE) is usually made by detecting specific antibodies in serum samples. However, collection of blood samples is difficult and may be hazardous and unsafe. Thus, it is important to assess alternative simple methods of sampling body fluids that give similar results. Saliva and urine have been suggested as possible alternatives to detect specific antibodies for the diagnosis of various diseases. To the best of our knowledge, there has been no previously published study regarding the detection of CE-specific immunoglobulin (Ig) G subclass antibodies (IgG1-4) in urine. Therefore, the present study was designed to assess the value of hydatid-specific antibodies of IgG, IgM, IgE and IgG subclass in urine and serum samples for the diagnosis of CE. Serum and urine samples of 41 surgically confirmed patients of CE, 40 patients with other diseases and 16 healthy subjects were included in the study. CE-specific total IgG, IgE and IgG4 in sera and total IgG, IgG4 and IgG1 in the urine of CE patients were the most important specific antibodies for the diagnosis of CE. However, total IgG usually persists for an extended period and has a very high cross-reactivity. The diagnostic sensitivity of hydatid-specific IgM in serum and urine samples was very low and therefore cannot be used as a diagnostic marker. There was no significant difference between IgG1 and IgG4 in serum and urine and both showed the best correlation for the diagnosis of CE. These considerations suggest that detection of antibodies in urine could provide a new approach in the diagnosis of CE.

Humoural immune response and pathological analysis in patients with false immune diagnosis of cystic echinococcosis.

The patients with false immune diagnosis of hydatid disease were investigated for the humoural immune response to analyse the possible reasons and mechanism leading to false immune diagnosis. Two hundred and thirty-nine patients with nature-unknown cysts and 30 healthy controls were detected by immunological assays (four hydatid antigen-based immunogold filtration assay and enzyme-linked immune absorbent assay) and ultrasound. Sensitivity of and specificity of immunological assay and ultrasound were calculated, respectively. The serological diagnosis was compared with surgical pathology to screen the patients with false immune diagnosis for the immunoglobulin measurement and pathological analysis. The history and cyst characteristics were also reviewed. The results indicate the immunoglobulin has little influence on false immunodiagnosis. The false-negative immunodiagnosis was caused by the cysts' inactive status while the false positive caused by previous rupture, antigen cross-reaction. The clinical diagnosis of cystic echinococcosis requires a combination of immunodiagnosis and ultrasonography, which is the necessary complementary confirmation.

Surgical management of liver hydatid disease: subadventitial cystectomy versus resection of the protruding dome.

BACKGROUND: The aim of this study was to compare postoperative outcome and long-term results after management of liver hydatid cysts (LHC) by subadventitial cystectomy (SC) and resection of the protruding dome (RPD) in two tertiary liver surgery centers. METHODS: Medical records of 52 patients who underwent SC in one center, and 27 patients who underwent RPD in another center between 1991 and 2011 were reviewed. Patients underwent long-term follow-up, including serology tests and morphological examinations. RESULTS: Postoperative mortality was nil. The rate of severe morbidity was 7.7 and 22% (p = 0.082), while the rate of serological clearing-up was 20 and 13.3% after SC and RPD, respectively (p = 1.000). After a mean follow-up of 41 months (1-197), four patients developed a long-term cavity-related complication (LTCRC) after RPD (including one recurrence) and none after SC (p = 0.012). All LTCRCs occurred in patients with hydatid cysts located at the liver dome; three required an invasive procedure by either puncture aspiration injection re-aspiration (N = 1) or repeat surgery (N = 2). CONCLUSIONS: RPD exposes to specific LTCRC, especially when hydatid cysts are located at the liver dome, while SC allows ad integrum restoration of the operated liver. Therefore, SC should be considered as the standard surgical treatment for LHC in experienced hepato-pancreato-biliary centers.

Pulmonary hydatid disease is difficult recognized in children.

PURPOSE: Pulmonary hydatid disease is a significant health problem for children in endemic areas. Pulmonary hydatid disease is more frequent than hepatic hydatid disease in children. The aim of this study was to evaluate the characteristics of pulmonary hydatid disease for children in our province which is endemic for echinococcosis. PATIENTS AND METHODS: This study was performed retrospectively between 2007 and 2012 by reviewing the medical records of patients (≤17 years) who were diagnosed with pulmonary hydatid disease. The medical records of these patients were evaluated with respect to the demographic characteristics of patients, management strategies, length of hospital stay, and outcomes. RESULTS: A total of 120 (52.9% boys and 46.3% girls) patients with the diagnosis of pulmonary hydatid disease were enrolled in this study. The mean age was 10.15 ± 3.93 years. The significant numbers of patients were admitted with a ruptured hydatid disease and managed with lung preservation. The mean follow-up was 11.3 ± 3.8 (3-24) months. Recurrence was detected in three patients during follow-up. CONCLUSION: Pulmonary hydatid disease is usually symptomatic. Hydatid disease must be considered in differential diagnosis while evaluating thoracic lesions in endemic areas.

Epidemic factors and control of hepatic echinococcosis in Qinghai province.

Hepatic echinococcosis, also called echinococcosis, is a health-threatening disease commonly found in pasture, and belongs to parasitic zoonoses. The purpose of this study was to investigate the prevalence and the risk factors of echinococcosis in Qinghai province in order to provide fundamental data for prevention and control of echinococcosis in Qinghai province. A total of 23 445 people from 21 counties were enrolled in this study by multi-stage stratified random sampling. Echinococcosis was diagnosed by using B-mode ultrasonography and serological tests. The results showed that the prevalence of echinococcosis was 4.47% (95%CI: 4.21%-4.73%) and serum positive rate (seroprevalence) was 15.47% (95%CI: 14.92%-16.02%) in 2010. The distribution of echinococcosis differed in age, sex, ethnicity, occupation and regions in Qinghai (P<0.05). GLMM analysis revealed that gender (female vs. male), ethnicity (Tibetan vs. other ethnicities), profession (herders vs. other professions) and region (autonomous prefectures vs. cities) were significant risk factors for echinococcosis (P<0.05). It was concluded that the prevalence of echinococcosis in 2010 was about 4% in Qinghai province, and the distribution of echinococcosis in Qinghai was associated with age, sex, ethnicity and profession.

[Ethological competence of Perm Territory dwellers in the assurance of personal infection safety].

The authors attempted to analyze preventive measures against infectious and parasitic diseases, which were used domestically by the Perm Territory population, their conjugacy with the stereotypes of attitude towards domestic animals, as well as behavioral features of compliance andcompetence in the assurance of infection safety. The found gaps in the assurance of personal infection safety (drinking unboiled water, unprotected sex, disregard of helminth prevention in domestic animals, and unwillingness to go in for sports) are coherent with the epidemiological situation in the Perm Territory and to our clinical and laboratory study of the patients of the Perm Territory Children's Clinical Hospital in 2011. Enzyme immunoassay (EIA) was used to examine 10075 patients for helminths and protozoa; parasitic diseases were detected in 2047 (20.3%) persons. The diagnostic titer of antibodies to Toxocara antigens was revealed in 677 (11.8%) children of 5700 patients examined for toxocariasis; that for Opisthorchis antigens was in 595 (37.7%) of 1578 examined for Opisthorchis infestation. The diagnostic titers for echinococcosis was found in 9 (0.75%) of 1198 patients; later on the diagnosis of hydatid disease was verified by epidemiological, clinical, and laboratory studies. Despite the inadequate informative value of EIA for the diagnosis of giardiasis, high antibody titers to Lamblia antigens were detected in 766 (47.9%) of 1599 children.

First case of Echinococcus vogeli infection imported to the Netherlands, January 2013.

In January 2013 in the Netherlands, a man in his 50s from Suriname underwent hemihepatectomy because of a cystic liver mass, assumed to be a cystadenoma. Pathology revealed an echinococcal infection. PCR analysis of cyst material identified Echinococcus vogeli, causing polycystic hydatid disease. This echinococcus species is rarely diagnosed outside South America. The patient received adequate treatment, but this case emphasises the importance of awareness of this infection when treating patients with cystic tumours from endemic areas.