Role of microRNAs in host defense against Echinococcus granulosus infection: a preliminary assessment.

Cystic echinococcosis (CE) is a neglected helminthic zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus s.l. MicroRNAs (miRNAs) are regulators of gene expression that have been linked with the pathogenesis of several human diseases, but little exists in the available literature about miRNAs in CE. Here, we investigate the expression profiles of 84 microRNAs relevant to the function of lymphocytes and other immune cells during CE infection in the peripheral blood of patients with cysts in active and inactive stages. We applied the microRNA PCR array technology to blood samples from 20 patients with a single hepatic CE cyst in either the active (CE3b) or inactive (CE4-CE5) stage. Our results show a significant upregulation of eight miRNAs (let-7g-5p, let-7a-5p, miR- 26a-5p, miR- 26b-5p, miR- 195-5p, miR- 16-5p, miR- 30c-5p, and miR- 223-3p) in patients with active cysts compared to those with inactive cysts. The high expression of these miRNAs in patients with active cysts suggests their role in a specific host immune response against the infection. Further work in this direction may help shed light on the pathogenesis of human CE.

Structural and Immunodiagnostic Characterization of Synthetic Antigen B Subunits From Echinococcus granulosus and Their Evaluation as Target Antigens for Cyst Viability Assessment.

Background: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.

[Cystic Echinococcosis Seropositivity in the Blood Samples Sent to Parasitology Laboratory of Yüzüncü Yil University Medical Faculty between 2005 and 2013: Retrospective Assessment]. / Yüzüncü Yil Üniversitesi Tip Fakültesi Parazitoloji Laboratuvarina 2005-2013 Yillari Arasinda Gönderilen Kan Örneklerinde Kistik Ekinokokkozis Seropozitifligi: Retrospektif Degerlendirme.

OBJECTIVE: This study was performed in order to contribute the data on the prevalence of cystic echinococcosis (CE) in Turkey and to reveal this parasitosis problem in Van province. METHODS: Blood samples of 2642 patients (1214 men, 1428 women; 506 of them 0-13 age, 2136 of them 14 and over age group), which were sent with suspected CE to Parasitology Laboratory from various policlinics of Yüzüncü Yil University Medical Faculty, between the dates of 01.09.2005 and 01.09.2013, were evaluated for CE by using Enzyme-Linked Immunosorbent Assay (ELISA) (R-Biopharm, Germany; IgG) method. RESULTS: 801 (30.3%) of examined blood samples of these patients were found positive for this parasitosis. Seropositivity was found 31.9% of men, 29% of women; 33.4% of 0-13 age group, 29.6% of 14 and over age group in the study. Cysts, which were obtained from operated seropositive patients, were confirmed that unilocular cysts as pathological. CONCLUSION: As a result, it was seen that CE is continued to be a major public health problem in Van province and it was appear that comprehensive protection and control programs should be carry out to struggle this disease.

Serodiagnosis of human hydatidosis with an ELISA developed based on antigens derived from sheep hydatid cysts and comparison with a commercial human ELISA kit.

OBJECTIVE: To explore the serodiagnosis of hydatid cyst in human using different antigens of sheep (hydatid fluid, Somatic and Excretory/secretory antigens of protoscolex) by ELISA and compares this result with commercial human ELISA kit. METHODS: One hundred blood samples from patients with history of severe abdominal pain and eosinophilia were obtained. Ten serum samples were obtained from surgically and pathologically confirmed cystic echinococcosis patients from Mashhad university hospital as positive control and 5 serum samples from infant under one year old as negative control. Blood samples were centrifuged at 3 000µg at 20 °C for 15 min and sera were stored at -20 °C. First, these samples were tested for the presence of antibody by commercial human ELISA. Then, ELISA was developed on microplates coated with hydatid fluid, Somatic and Excretory/secretory antigens of protoscolex of sheep. RESULTS: The results of this study as analyzed by Kappa test showed that, hydatid fluid antigen could be used as a precise source of detection in indirect ELISA test. CONCLUSIONS: Hydatid fluid in comparison with Excretory-secretory and somatic antigens showed more compatibility agreement in kappa test which can be used for further studies in development of any ELISA test for diagnosis of human hydatidosis.

Isolation and molecular characterization of recombinant Echinococcus granulosus P29 protein (recP29) and its assessment for the post-surgical serological follow-up of human cystic echinococcosis in young patients.

We synthesized recombinant Echinococcus granulosus protoscolex recP29 antigen to be preliminarily assessed by ELISA and immunoblotting. RecP29-serology was carried out on 54 young patients with cystic echinococcosis (CE). Patients were classified into either cured (CCE) (n=40) or non-cured (NCCE) (n=14) CE patients. RecP29 ELISA showed a gradual decrease of antibody concentrations in all CCE cases that were initially (before treatment) seropositive to this antigen (25 out of 40) or that seroconverted following treatment. A complete seronegativity was reached within 3 years post-surgery in all of these cases. Conventional HCF ELISA yielded seronegativity in only 10% of initially recP29-seropositive CCE patients (P=0.086). Likewise, recP29 immunoblotting yielded seronegativity in 93% of 29 out of 40 initially recP29-immunoblot-positive CCE patients after 3 years follow-up, compared with 72% in the HCF immunoblotting (P=0.060). Eleven out of 14 NCCE patients were initially positive by recP29 ELISA, and 10 out of these maintained a marked anti-recP29 antibody reactivity until the endpoint of the follow-up period. All 14 NCCE cases were initially seropositive by recP29 immunoblotting, and 13 cases remained seropositive until the end of the study. Thus, recombinant P29 protein appears prognostically useful for monitoring those post-surgical CE cases with an initial seropositivity to this marker.

Assessment of in vivo complement activation on the Echinococcus granulosus hydatid cyst wall.

The larval stage of the parasite Echinococcus granulosus causes hydatid disease. The hydatid cyst is potentially capable of activating host complement, since it is a large, persistent, carbohydrate-rich structure, coated with host immunoglobulins, and localized in the host's internal organs. Nonetheless, in vitro studies have suggested that the cyst surface, the hydatid cyst wall (HCW), is a poor complement activator. In this study, we assessed the occurrence of in vivo complement activation on the hydatid cyst by measuring the levels of two complement activation products, C3d and complexes bearing a C9 activation neoepitope (TCC/MAC), in extracts from HCW of human origin. Low amounts of C3d and TCC/MAC were found in HCW in comparison with their levels in normal human plasma and activated human sera, suggesting that in vivo complement activation on HCW is efficiently down-regulated. This regulation may contribute to limit host inflammation which has been observed to correlate with parasite degeneration and death.

Assessment of protective immune responses against hydatid disease in sheep by immunization with synthetic peptide antigens.

Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

Assessment of the immunological surveillance value of humoral and lymphocyte assays in severe human cystic echinococcosis.

Thirty cystic echinococcosis (CE) patients in Uruguay with severe bone or secondary disseminated echinococcosis were immunologically assessed using cellular (lymphocyte transformation assay, LTA) and humoral (specific antibody and subclass responses, circulating antigen and circulating immune-complexes) immunological assays during the course of chemotherapy (albendazole and/or praziquantel). CE patients were divided into 4 groups, according to clinical treatment and outcome: (I) surgery and chemotherapy, (II) chemotherapy with outcome unchanged, (III) chemotherapy with outcome improved, and (IV) chemotherapy considered cured. Increased circulating antigen was of prognostic value in some severe CE cases where levels remained high and/or increased. The lymphoproliferative response in vitro to Echinococcus granulosus antigen was statistically greater in all patient groups compared to normal individuals but at lower levels in improved or cured CE patients. Levels of non-specific LTA response were significantly lower than controls for all groups during albendazole treatment (P < 0.001) but returned to normal levels in cured patients, a result consistent with parasite-induced suppression of cellular responses. This study suggests that, at least in severe osseous and secondary CE, immunosurveillance by specific antibodies, especially total specific immunoglobulin, was overall of more practical use than antigen-specific in-vitro lymphocyte transformation assays.

Immunological assessment of exposure to Echinococcus granulosus in a rural dog population in Uruguay.

An ELISA was used to screen a dog population in Uruguay (Sarandi Del Yi, Durazno District) for the prevalence of specific serum antibodies (IgG, IgA and IgE) to Echinococcus granulosus. The sensitivity (61%) and specificity (97%) of the ELISA were determined using well-defined serum groups. A total of 408 dogs from Sarandi del Yi and environs were screened serologically, and 29.7% (8.6-13.8% for each antibody class) of dogs had positive levels of antibody to E. granulosus. This antibody prevalence (exposure) was significantly higher than the percentage of dogs found to be positive for E. granulosus worms by arecoline purgation (7.6%). This level of exposure to E. granulosus determined by ELISA is considered unacceptable from a public health perspective. Measures will now focus on obtaining data on the true prevalence of current infection in this dog population and on determining the transmission patterns of the disease in this endemic region.

Some observations on hydatidosis in Jordan.

A total of 2.8% (5 of 176) sera from schoolchildren and university students were seropositive for hydatidosis (Echinococcus granulosus), by the ELISA IgE test. The prevalence of seropositivity was 4.5% among females of all age groups (5-24 years) and among males of the age group 18-24 years. Males of the age group (5-17 years) were seronegative. The ratio of seropositivity between schoolboys and schoolgirls was 1:4. Sera collected from 1085 university students, aged 18-24 years, and from 1656 outpatients from northern and central Jordan, and the Jordan River valley, when tested by the IHA test, revealed the presence of anti-hydatid agglutinating antibodies in 5.16% (56 of 1085) and in 3.6% (60 of 1656) respectively, with a male to female infection ratio of 1:3 in the university students and 1:1.1 in the outpatients. Intensity, prevalence and cyst fertility of E. granulosus increased in sheep with age. The results of this study indicate that hydatidosis is in an endemic steady state in Jordan with estimated economic losses of JD 3.0 (= US $4.4) per infected sheep.

Assessment of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B for detection of human hydatid circulating antigens.

Four monoclonal antibodies (MAb) to Echinococcus granulosus Antigen 5 (Ag5) and Antigen B (AgB) were assessed in an enzyme-linked immunosorbent assay (ELISA) for detection of circulating antigens (CAg) in sera of human patients with E. granulosus infection. Around 5.5-8% of 200 sera from 42 surgically proven hydatid patients contained detectable CAg by individual MAb. The combined detection rate for CAg, using four MAb, was 19% (38/200). Although hydatid CAg was detected by MAb in at least one serum sample from 21 of 42 patients, some patients remained negative in the assay regardless of the time when serum samples were taken (pre- or post-operatively), or of the continuing presence of hydatid cysts, their location or fertility. In addition, it was observed that the binding capacity of MAb for sheep hydatid cyst fluid antigen (SHCF) was somewhat reduced in the presence of normal human serum. The CAg detection assay would only be useful for assessment of hydatid infection status in patients with detectable CAg in serum samples.

[The assessment of the epidemiologic situation of echinococcosis in the Nenets Autonomous Okrug based on data from a dynamic serologic examination of the population]. / Otsenka épidemiologicheskoi situatsii po ékhinokokkozam v Nenetskom avtonomnom okruge po dannym dinamicheskogo serologicheskogo obsledovaniia naseleniia.

Paper presents the RIHA and EIA data of an annual 3-year survey of populations of two reindeer breeders' settlements in the Nenets Autonomous Okrug of Arkhangelsk Province with the use of intact Echinococcus antigen (RIHA) and fractions of Echinococcus multilocularis antigens (EIA). In 1984-1986, seropositivity has been established by 2 immunoassays for 98 (21.6 +/- 1.9%) out of 454 apparently healthy subjects. The annual Echinococcus invasion rate in population varied within 2.8-3.7%, as estimated by RIHA, and 4.6-4.8%, according to EIA data. The highest invasion rate was observed among the those engaged in reindeer breeding, hunting, animal breeding as well as among the aboriginal populations the Nentsi and the Komi.