High Expression of Tim-3 in Alveolar Echinococcosis Mediates Depletion of CD8+ T Cell Function.

OBJECTIVE: CD8+ T cells can participate in immune action by secreting various cytokines, which have a killing effect on certain viruses, tumor cells, and other antigenic substances. However, in studies such as chronic viral infections and some parasitic infections, CD8+ T lymphocyte showed functional depletion, and its immune dysfunction was an important reason for the persistence of infection. Tim-3 has been shown to be a negative regulator of CD8+ T cell function, causing depletion of CD8+ T cells in cancer and chronic infection. However, the relationship between Tim-3 and CD8+ T cells in Echinococcus multilocularis infection is not clear. METHODS: In this study, we analyzed peripheral blood CD8+ T cells from 62 alveolar echinococcosis (AE) patients and 30 healthy controls. RESULTS: Compared with the healthy control group, the proportion of CD8+ T cells in the peripheral blood of AE patients increased significantly, while the levels of perforin, granzyme B and IFN-γ in peripheral blood CD8+ T cell related factors of metabolically active alveolar echinococcosis (MAAE) patients decreased significantly. Later detection revealed that the expression of Tim-3 on CD8+ T cells in the peripheral blood of MAAE patients was significantly higher than that of metabolically inactive alveolar echinococcosis (MIAE) patients and healthy controls. The expression levels of function-related factors perforin, granzyme B and IFN-γ in CD8+ Tim-3+ T cell were significantly lower in the CD8+Tim-3- T cells of AE patients. In vitro, the secretion of CD8+ T cell-associated factors was significantly restored by inhibiting Tim-3 expression. CONCLUSION: Therefore, the depletion of CD8+ T lymphocyte in patients with alveolar echinococcosis disease is considered to be related to the high expression of Tim-3 on the surface.

Molecular characterization and immune protection of the 3-hydroxyacyl-CoA dehydrogenase gene in Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (CE) is a serious parasitic zoonosis caused by the larvae of the tapeworm Echinococcus granulosus. The development of an effective vaccine is one of the most promising strategies for controlling CE. METHODS: The E. granulosus 3-hydroxyacyl-CoA dehydrogenase (EgHCDH) gene was cloned and expressed in Escherichia coli. The distribution of EgHCDH in protoscoleces (PSCs) and adult worms was analyzed using immunofluorescence. The transcript levels of EgHCDH in PSCs and adult worms were analyzed using quantitative real-time reverse transcription PCR (RT-qPCR). The immune protective effects of the rEgHCDH were evaluated. RESULTS: The 924-bp open reading frame sequence of EgHCDH, which encodes a protein of approximately 34 kDa, was obtained. RT-qPCR analysis revealed that EgHCDH was expressed in both the PSCs and adult worms of E. granulosus. Immunofluorescence analysis showed that EgHCDH was mainly localized in the tegument of PSCs and adult worms. Western blot analysis showed that the recombinant protein was recognized by E. granulosus-infected dog sera. Animal challenge experiments demonstrated that dogs immunized with recombinant (r)EgHCDH had significantly higher serum IgG, interferon gamma and interleukin-4 concentrations than the phosphate-buffered saline (PBS) control group. The rEgHCDH vaccine was able to significantly reduce the number of E. granulosus and inhibit the segmental development of E. granulosus compared to the PBS control group. CONCLUSIONS: The results suggest that rEgHCDH can induce partial immune protection against infection with E. granulosus and could be an effective candidate for the development of new vaccines.

Design of a Novel Multi-Epitope Vaccine Against Echinococcus granulosus in Immunoinformatics.

All the time, echinococcosis is a global zoonotic disease which seriously endangers public health all over the world. In order to speed up the development process of anti-Echinococcus granulosus vaccine, at the same time, it can also save economic cost. In this study, immunoinformatics tools and molecular docking methods were used to predict and screen the antigen epitopes of Echinococcus granulosus, to design a multi-epitope vaccine containing B- and T-cell epitopes. The multi-epitope vaccine could activate B lymphocytes to produce specific antibodies theoretically, which could protect the human body against Echinococcus granulosus infection. It also could activate T lymphocytes and clear the infected parasites in the body. In this study, four CD8+ T-cell epitopes, three CD4+ T-cell epitopes and four B-cell epitopes of Protein EgTeg were identified by immunoinformatics methods. Meanwhile, three CD8+ T-cell epitopes, two CD4+ T-cell epitopes and four B-cell epitopes of Protein EgFABP1 were identified. We constructed the multi-epitope vaccine using linker proteins. The study based on the traditional methods of antigen epitope prediction, further optimized the prediction results combined with molecular docking technology and improved the precision and accuracy of the results. Finally, in vivo and in vitro experiments had verified that the vaccine designed in this study had good antigenicity and immunogenicity.

Accuracy of an experimental whole-blood test for detecting reactivation of echinococcal cysts.

BACKGROUND: Cystic echinococcosis (CE) is a complex disease for which clear understanding of clinical manifestations is needed to avoid misdiagnosis, inappropriate treatment, and severe complications. We evaluated the accuracy of a whole-blood stimulation test based on Interleukin (IL)-4 detection in response to Antigen B (AgB) of Echinococcus granulosus sensu lato to discriminate cyst viability and detect cyst reactivation in patients with hepatic CE. METHODOLOGY/PRINCIPAL FINDINGS: Thirty patients with CE3b cysts and 37 patients with spontaneously-inactivated CE4-CE5 cysts were recruited (T0). After enrollment, 5 patients with CE3b cysts received albendazole, resulting in cyst solidification (CE4) in 4/5. Within a two-year follow-up, the whole-blood test was repeated at two time-points, in ≥14 (T1) and in ≥4 (T2) patients per group. IL-4 and a panel of other soluble factors were measured in the stimulated plasma. Baseline IL-4 levels were significantly higher in patients with CE3b compared to those with CE4 cysts (p = 0.006). Test accuracy for CE3b diagnosis had a sensitivity of 33-60% and a specificity of 76-95%, depending on the cut-off applied. Overall, IL-4 levels did not change significantly over time in either group; however, patients within the CE3b group showed a significant decrease of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF at T1 compared to T0 (p≤0.042). CONCLUSIONS/SIGNIFICANCE: Whole-blood IL-4-response to AgB is significantly higher in patients with active compared to inactive CE but apparently not modulated over time after treatment. On the contrary, the levels of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF significantly decreased in active CE during follow-up. Additional studies are needed to understand whether these findings might have a clinical significance for patients' follow-up.

Serologic evidence of Echinococcus granulosus in slaughterhouses in Pakistan: global alarm for butchers in developing countries.

INTRODUCTION: Cystic echinococcosis, caused by Echinococcus granulosus, is a neglected zoonosis that affects humans and livestock. This sero-survey was designed for the first time in Pakistan to assess the exposure of butchers to E. granulosus as there was no previous report in the country for this occupational group. METHODOLOGY: Blood samples were collected from registered butchers (n = 364) in five different slaughterhouses in Faisalabad and Bahawalnagar Districts. Sera were tested for anti-Echinococcus granulosus IgG with a commercially available ELISA kit (specificity, 100%; sensitivity, 97%). RESULTS: Overall, seroprevalence was 9.61% (35/364). Butchers >30 years of age (10.34%), those involved in small ruminants butchery (11.70%), >10 years' experience (10.04%), formal education level up to middle standard (10.28%), contact with dogs (12.71%), improper/unhygienic disposal of dog feces (11.87%), and those unaware of the consequences of eating with unwashed hands (13.80%) were more seropositive with significant statistical differences (p < 0.05). Variables like previous cyst encounter, no knowledge of zoonoses and/or cystic echinococcosis, living in rural areas and the presence of stray/feral dogs in surroundings did not show any significant association (p > 0.05) with seroprevalence in butchers. The binary logistic regression model also showed a statistically significant relationship (p < 0.05) for all risk factors found statistically significant (p < 0.05) in the univariate analysis. CONCLUSIONS: This study shows high prevalence of cystic echinococcosis among butchers in Pakistan and underscores the need for educating native slaughterhouse personnel on cystic echinococcosis. It also serves as a global warning, especially in developing countries.

microRNA-125b-5p is a promising novel plasma biomarker for alveolar echinococcosis in patients from the southern province of Qinghai.

BACKGROUND: Alveolar echinococcosis (AE) is caused by parasitic infection by Echinococcus multilocularis. Its diagnosis is usually based on clinical symptoms, ultrasound, and other imaging methods. MicroRNAs (miRNAs) play important roles in disease processes and can exist in a highly stable cell-free form in body fluids. It is important to identify specific, sensitive diagnostic markers for early diagnosis and evaluation of AE. In this study, we examined hsa-miR-125b-5p as a potential plasma biomarker of E. multilocularis infection. METHODS: Plasma samples from patients with AE and healthy individuals were screened for the presence of five miRNAs using miRNA chips. We used quantitative polymerase chain reaction to measure miRNA expression levels in plasma and liver tissue samples from patients with AE. RESULTS: hsa-miR-125b-5p was stably upregulated in the plasma and liver tissue samples from patients with AE. CONCLUSIONS: The results suggest that hsa-miR-125b-5p may be a promising biomarker for early, non-invasive diagnosis of AE.

Promising proteins detected by Western blot from Echinococcus granulosus protoscoleces for predicting early post-surgical outcomes in CE-affected Tunisian children.

BACKGROUND: Cystic echinococcosis (CE) affects predominantly young patients in highly endemic areas. Improved serological methods are needed for the follow-up of CE cases, especially given the high rates of post-surgical relapse that require detection as soon as possible. METHODS: We designed a study to investigate the value of antigenic proteins extracted from Echinococcus granulosus (E. granulosus) protoscoleces, and of recombinant B2t and 2B2t proteins, for assessing the efficacy of surgical treatment carried out on CE-affected children. This study was performed on 278 plasma samples collected from 59 Tunisian children surgically treated for CE and monitored for 3 years post-surgery. The patients were classified according to post-surgical outcomes into a "non-relapsed" (NRCE) and a "relapsed" (RCE) group. We performed in-house ELISAs to measure anti-B2t and anti-2B2t IgG and immunoblotting for the detection of IgG against SDS-PAGE-resolved E. granulosus protoscoleces-specific antigens. The Wilcoxon test was applied to assess anti-B2t and anti-2B2t IgG levels. We applied the Cochran Q test to compare the distribution of immunoblotting antigenic bands between 1-month and 1-year post-surgery. RESULTS: The probability of being "relapse-free" when a decrease in antibody titers occurred between 1 month and 1 year post-surgery was 81% and 75%, respectively, for anti-B2t and anti-2B2t IgG. We identified five protoscolex protein bands of 20, 26/27, 30, 40 and 46 kDa as highly immunoreactive by immunoblot for both RCE and NRCE patients at 1 month post-surgery, and significantly lower immunoreactivity after 1 year (p < 10-4) for NRCE compared to RCE patients. The proteins at 26/27 and 40 kDa displayed the best performance in predicting the outcome, with an 84% probability of being relapse-free when the reactivity against the 40 kDa antigen, the doublet at 26/27 kDa, or both was absent or disappeared between 1 month and 1 year post-surgery, and a 93% probability of being relapsed when both bands remained reactive or increased in intensity between the two time points. CONCLUSIONS: The B2t protein could be useful for the prediction of CE early post-surgical outcomes. The proteins of E. granulosus protoscoleces, especially the doublet P26/27 and P40, could be promising predictive biomarkers for the post-surgical follow-up of CE cases as well.

Interleukin 17 Cytokine Profiles in Patients with Cystic Echinococcosis in Babylon Province, Iraq.

Cystic Echinococcosis (CE) is the silent chronic Helminthes zoonotic infection caused by the larval stage in intermediate hosts of the dog tapeworm Echicoccous granulosus, which belongs to the Taeniidae family and genus Echinococcu ssp. According to the study of CE, the hospitalization and surgeries of patients indicated the high prevalence of the disease in Iraq.This study aimed to determine some immunological parameters in patients infected with Echinococcus granulose. The study of 53 patients infected with CE showed that in 40 (75.4 %), 5 (9.4%), 4 (7.5%), and 2 (3.7 %) cases the liver, abdominal cavity, kidney, and lungs were involved. In terms of age, most and least number of the patients were within the age ranges of30-40 (n=15, 28.3 %) and60-70 years old (n=7, 13.2 %), respectively. Moreover, 37(56.9%) and 16(43%) of them were female and male, respectively. The enzyme-linked immunosorbent assay (ELISA) was used to measure the level of the Interleukin family among patients. There was a significant increase in the serum level concentration of IL17A and IL17B in patients with hydatid disease, compared to the control group. The changes in different age groups also showed statistically significant differences among them (P≤0.05). The outcome of this study indicated that CE is endemic in Babylon province, Iraq. The ELISA technique is a reliable and efficient test for the early diagnosis and monitoring of human hydatid disease. Moreover, it was found that the liver was the most common site of human hydatid cyst.

Characterizing dynamic changes of plasma cell-free Echinococcus granulosus DNA before and after cystic echinococcosis treatment initiation.

Over one million people are living with cystic echinococcosis (CE) and alveolar echinococcosis (AE). For CE, long-term albendazole treatment is often needed, which requires regular follow-up. Follow-up is mainly through imaging which is insensitive to subtle changes and subjective to experience. We investigated the changes of Echinococcus granulosus (Eg) cell-free DNA (cfDNA) in plasma of CE patients before and after albendazole treatment to evaluate its potential as an objective marker for treatment follow-up. Plasma samples of nine CE patients were collected before and after treatment. We identified Eg cfDNA from every sample through high-throughput sequencing. Eg cfDNA concentration and fragment length increased significantly after the treatment period. Ultrasound examination before and after the treatment initiation reflected the drug effects to a certain extent, as the cyst size of four patients reduced. Our findings indicated that Eg cfDNA from plasma could be a potential marker in the monitoring of CE treatment.

Retrospective Evaluation of Cases with the Pre-diagnosis of Cystic Echinococcosis Admitted to the Microbiology Laboratory of Diyarbakir Health Sciences Gazi Yasargil Training and Research Hospital Between 2014 and 2017.

OBJECTIVE: Cystic echinococcosis (CE) is a zoonotic disease, which leads to morbidity and mortality worldwide. This study aimed to retrospectively evaluate the presence of anti-Echinococcus granulosus immunoglobulin G (IgG) antibodies, which were detected by indirect fluorescent antibody test in the samples that were transferred to the Microbiology Laboratory of University of Health Sciences Turkey, Diyarbakir Gazi Yasargil Training and Research Hospital with the pre-diagnosis of CE. Moreover, gender differences with respect to positivity rates of anti-E. granulosus IgG antibodies were investigated. METHODS: Anti-E. granulosus IgG antibodies, which were detected in the samples of cases with the pre-diagnosis of CE between January 2014 and December 2017, were retrospectively evaluated. Gender difference with respect to positivity rates was investigated by applying the chi-square test in cases with positive anti-E. granulosus IgG antibodies. RESULTS: Out of the 829 serum samples, 222 (26.7%) were found to be positive for E. granulosus IgG antibodies, among which 40 (27.2%), 56 (25.5%), 51 (23.3%) and 75 (30.6%) were found to be positive in 2014, 2015, 2016 and 2017, respectively. No significant difference was found between genders amongst the cases with positive anti-E. granulosus IgG antibodies (p>0.05). CONCLUSION: Since CE is a major public health problem, evaluation of the presence of anti-E. granulosus IgG antibodies would be important to understand the positivity rate at the regional level.

Proteomic analysis of plasma exosomes from Cystic Echinococcosis patients provides in vivo support for distinct immune response profiles in active vs inactive infection and suggests potential biomarkers.

The reference diagnostic method of human abdominal Cystic Echinococcosis (CE) is imaging, particularly ultrasound, supported by serology when imaging is inconclusive. However, current diagnostic tools are neither optimal nor widely available. The availability of a test detecting circulating biomarkers would considerably improve CE diagnosis and cyst staging (active vs inactive), as well as treatments and follow-up of patients. Exosomes are extracellular vesicles involved in intercellular communication, including immune system responses, and are a recognized source of biomarkers. With the aim of identifying potential biomarkers, plasma pools from patients infected by active or inactive CE, as well as from control subjects, were processed to isolate exosomes for proteomic label-free quantitative analysis. Results were statistically processed and subjected to bioinformatics analysis to define distinct features associated with parasite viability. First, a few parasite proteins were identified that were specifically associated with either active or inactive CE, which represent potential biomarkers to be validated in further studies. Second, numerous identified proteins of human origin were common to active and inactive CE, confirming an overlap of several immune response pathways. However, a subset of human proteins specific to either active or inactive CE, and central in the respective protein-protein interaction networks, were identified. These include the Src family kinases Src and Lyn, and the immune-suppressive cytokine TGF-ß in active CE, and Cdc42 in inactive CE. The Src and Lyn Kinases were confirmed as potential markers of active CE in totally independent plasma pools. In addition, insights were obtained on immune response profiles: largely consistent with previous evidence, our observations hint to a Th1/Th2/regulatory immune environment in patients with active CE and a Th1/inflammatory environment with a component of the wound healing response in the presence of inactive CE. Of note, our results were obtained for the first time from the analysis of samples obtained in vivo from a well-characterized, large cohort of human subjects.

Cattle co-infection of Echinococcus granulosus and Fasciola hepatica results in a different systemic cytokine profile than single parasite infection.

E. granulosus is a cestode that causes Cystic Echinococcosis (CE), a zoonotic disease with worldwide presence. The immune response generated by the host against the metacestode induces a permissive Th2 response, as opposed to pro-inflammatory Th1 response. In this view, mixed Th2 and regulatory responses allow parasite survival. Overall, larval Echinococcus infections induce strong regulatory responses. Fasciola hepatica, another common helminth parasite, represents a major infection in cattle. Co-infection with different parasite species in the same host, polyparasitism, is a common occurrence involving E. granulosus and F. hepatica in cattle. 'While it is known that infection with F. hepatica also triggers a polarized Th2/Treg immune response, little is reported regarding effects on the systemic immune response of this example of polyparasitism. F. hepatica also triggers immune responses polarized to the Th2/ Treg spectrum. Serum samples from 107 animals were analyzed, and were divided according to their infection status and Echinococcal cysts fertility. Cytokines were measured utilizing a Milliplex Magnetic Bead Panel to detect IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-18. Cattle infected only with F. hepatica had the highest concentration of every cytokine analyzed, with both 4.24 and 3.34-fold increases in IL-10 and IL-4, respectively, compared to control animals, followed by E. granulosus and F. hepatica co-infected animals with two-fold increase in IL-10 and IL-4, compared to control animals, suggesting that E. granulosus co-infection dampens the cattle Th2/Treg immune response against F. hepatica. When considering Echinococcal cyst fertility and systemic cytokine concentrations, fertile cysts had higher IFN-γ, IL-6 and IL-18 concentrations, while infertile cysts had higher IL-10 concentrations. These results show that E. granulosus co-infection lowers Th1 and Th2 cytokine serological concentration when compared to F. hepatica infection alone. E. granulosus infections show no difference in IFN-γ, IL-1, IL-2, IL-6 and IL-18 levels compared with control animals, highlighting the immune evasion mechanisms of this cestode.

Results of Radiological and Serological Screenings in Individuals Sharing the Same Living Area as Patients with Hydatid Cyst in Afghanistan's State Hospital

Objective: To determine the results of radiological and serological screenings in individuals who shared the same living space as patients with hydatid cyst in a State Hospital of Afghanistan. Methods: Patients presenting with hydatid cyst to a public hospital in Afghanistan were included in this study. Full sampling method was used. Also, the relatives of the patients were called to the hospital and investigated for the presence of hydatid cyst antibodies through direct chest X-ray, upper abdominal ultrasonography and ELISA. Results: During the study period, a total of 214 patients, including 102 male and 112 female, underwent radiological and serological screenings. While cysts were radiologically detected in the liver, lung and spleen in 8, 2 and 1 patient, respectively, the serology was positive in 22 patients. Conclusion: As a result of the study, it was concluded that the patients who shared the same environment as the patients who were diagnosed with hydatid cyst in an endemic region for cyst hydatid disease.

Serodiagnosis of human cystic echinococcosis based on recombinant antigens B8/1 and B8/2 of Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (CE) is a widespread parasitic disease caused by the larval stage of Echinococcus granulosus. Since current methods for the diagnosis of CE are not efficient enough, rapid, and reliable tests are required for the acceleration of CE diagnosis. The present study aimed to produce recombinant B8/1 and B8/2 antigens of E. granulosus and evaluate their sensitivities and specificities separately and simultaneously for the diagnosis of CE. METHODS: The recombinant B8/1 and B8/2 antigens were produced and used in an ELISA system for the diagnosis of CE. The sera specimens including 30 sera from pathologically confirmed CE patients, 30 from other non-CE patients, and 30 from healthy controls, were evaluated by the ELISA, using AgB8/1 and AgB8/2. RESULTS: The results showed a sensitivity of 93.33%, 90%, and 96.7% for AgB8/1, AgB8/2, and their combination, respectively. The specificities were 91.7%, 93.33%, and 93.33% for AgB8/1, AgB8/2, and their combination, respectively. CONCLUSION: Simultaneous usage of AgB8/1 and AgB8/2 increased the test sensitivity for the diagnosis of CE. Furthermore, the specificity of AgB8/1 and AgB8/2 combination was more than AgB8/1 and equal to AgB8/2 alone. The findings revealed that the simultaneous usage of AgB8/1 and AgB8/2 could be a suitable approach for the diagnosis of CE.

The correlations between Th1 and Th2 cytokines in human alveolar echinococcosis.

BACKGROUND: Alveolar echinococcosis (AE) is a zoonotic parasitic disease caused by Echinococcus multilocularis larval tapeworm infections in humans that severely impairs the health of affected patients in the northern hemisphere. METHODS: The expression levels of 20 cytokines associated with AE infection were measured by enzyme-linked immunosorbent assay, and the correlations between these cytokines were analysed in the R programming language. RESULTS: Serum cytokine levels differed among individuals in both the AE patient and healthy control groups. The results of the correlations among the cytokines showed obvious differences between the two groups. In the AE patients group, Th1 and Th2 cytokines formed a more complicated network than that in the healthy control group. CONCLUSIONS: The altered correlations between Th1 and Th2 cytokines may be closely associated with AE infection, which may provide a new explanation for the essential differences between AE patients and healthy individuals.

Diagnostic performance of Echinococcus granulosus protoscolices antigens in the serodiagnosis of human cystic echinococcosis.

The development of suitable serological tests for the diagnosis of CE is still necessary. This study aimed to evaluate the efficacy of ELISA in the diagnosis of human cystic echinococcosis (CE), using parasite protoscolices antigens. Liver hydatid cysts were isolated from sheep infected with hydatid cysts and the protoscolices were isolated from the hydatid cyst fluid. Protoscolices crude antigen was prepared by mechanical disruption, plus freeze-thawing and sonication methods. Thirty sera samples of confirmed hydatid cyst patients, 30 samples of healthy individuals, and 30 samples of people with other infections were collected and the samples were evaluated in an ELISA system, using the crude protoscolices antigen. The sera samples were also simultaneously evaluated by antigen B-ELISA. The estimated value of sensitivity and specificity for the ELISA, using the crude protoscolices antigens, was 93.3% (95% CI: 76.4-98.8%) and 90% (95% CI: 78.8-95.8%), respectively. These values were 86.6 (95% CI: 68.3-95.6) and 91 (95% CI: 80.81-96.9) for the antigen-B based ELISA. Antigens prepared from protoscolices of hydatid cyst are suitable candidates for the serologic diagnosis of human CE. Further studies are needed to identify a single specific antigen among the protoscolices antigens to improve the diagnostic performance of these antigens.

Echinococcosis with the image of peritoneal carcinomatosis.

OBJECTIVE: Describe the case of echinococcal disease in gynecological practice and point out the complications of its diagnosis. DESIGN: Case report. SETTING: Department of Obstetrics and Gynaecology, Faculty Hospital Trenčín, Slovakia. CASE REPORT: The case is presented in an immunosuppressed patient with peritoneal carcinomatosis, mild Ca 125 elevation and increased CRP presenting as a finding of gynecological etiology with histopathological outcome and conclusion of parasitic disease - echinococcosis (hydatidosis). CONCLUSION: In the differential diagnosis of peritoneal carcinomatosis and ascites, especially in immunosuppressed patients with a positive social (or epidemiological) history, the possibility of rare parasitic diseases such as echinococcosis, which resembles malignant tumors, should be considered.

Rapid and label-free screening of echinococcosis serum profiles through surface-enhanced Raman spectroscopy.

Echinococcosis is a serious zoonotic parasitic disease that could be fatal without diagnosis and treatment in a timely manner. Herein, we present a rapid and label-free method for screening of echinococcosis using surface-enhanced Raman spectroscopy (SERS). Three groups of serum SERS spectra based on porous silicon/silver composites are obtained: one group from healthy volunteers (normal, n = 163) and two other groups from patients with pathologically confirmed echinococcosis (cystic echinococcosis (CE), n = 69 and alveolar echinococcosis (AE), n = 38). The derived characteristic spectrum was analyzed to explain differences between echinococcosis and healthy volunteers and a principal component analysis (PCA) was applied for classification. Raman spectra revealed that high sensitivity and specificity for echinococcosis diagnosis were associated with the contents of phenylalanine and tyrosine. In addition, Raman spectroscopy analysis identified two metabolites including phenylalanine and carotenoids that could distinguish three types of serum. Orthogonal partial least squares discriminant analysis (OPLS-DA) was successfully used as a discriminative model to classify echinococcosis with the highest sensitivity and specificity of 100% and 98.6%, respectively. Combination of serum metabolomics with SERS enabled accurate screening of echinococcosis patients. The results indicate that SERS-based serum profile analysis has the potential to be a valuable tool for the early diagnosis and screening of echinococcosis.

Parasite-derived microRNAs in plasma as novel promising biomarkers for the early detection of hydatid cyst infection and post-surgery follow-up.

Currently, cystic echinococcosis (CE) follow-up is a serious concern among surgeons. MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs which are present in human body fluids in a highly stable form. Recently, it is observed that Echinococcus granulosus expresses a large number of miRNAs in its developmental stages. The current study aimed at evaluating the capacity of parasitic miRNAs to serve as plasma biomarkers for hydatid cysts before and after CE surgery. Hydatidosis patients were identified using radiological and histopathological examinations. Following RNA extraction and cDNA synthesis, the expression levels of parasite-derived miRNAs including egr-miR-71 and egr-let-7 were quantitatively evaluated using real-time polymerase chain reaction (RT-PCR) in 30 hydatid cyst-infected individuals before surgery and an equal number of healthy controls. Then, three- and six-month follow-ups were performed after cystectomy. To analyze parasite-derived miRNAs, the relative fold change between uninfected and infected samples was determined and normalized to hsa-miR-16-5p as the housekeeping internal control. RT-PCR demonstrated that egr-miR-71 and egr-let-7 were specifically amplified in all the plasma samples from the infected individuals with hydatid cyst; yet they were significantly down-regulated at three and six months' post-surgery (P < 0.05). The egr-miR-71 had a higher level of expression in larval stage compared with egr-let-7. The results of the current study indicated that hydatid cyst-derived miRNAs including egr-miR-71 and egr-let-7 can be detected in human plasma. Considering the changes in the expression levels of these miRNAs after three and six months, it seems that these miRNAs, especially egr-miR-71, could serve as novel promising biomarkers for the early diagnosis and monitoring of hydatidosis.

Proteomic Analysis on Exosomes Derived from Patients' Sera Infected with Echinococcus granulosus.

Cystic echinococcosis (CE), a zoonotic disease caused by Echinococcus granulosus at the larval stage, predominantly develops in the liver and lungs of intermediate hosts and eventually results in organ malfunction or even death. The interaction between E. granulosus and human body is incompletely understood. Exosomes are nanosized particles ubiquitously present in human body fluids. Exosomes carry biomolecules that facilitate communication between cells. To the best of our knowledge, the role of exosomes in patients with CE is not reported. Here, we isolated exosomes from the sera of patients with CE (CE-exo) and healthy donors and subjected them to liquid chromatography-tandem mass spectrometry analysis. Proteomic analysis identified 49 proteins specifically expressed in CE-exo, including 4 proteins of parasitic origin. The most valuable parasitic proteins included tubulin alpha-1C chain and histone H4. And 8 proteins were differentially regulated in CE-exo (fold change>1.5), as analyzed with bioinformatic methods such as annotation and functional enrichment analyses. These findings may improve our understanding about the interaction between E. granulosus and human body, and may contribute to the diagnosis and prevention of CE.