Primary pelvic Echinococcus granulosus infection:A case report

BACKGROUND Primary pelvic Echinococcus granulosus infection is clinically rare.The reported cases of pelvic Echinococcus granulosus infection are considered to be secondary to cystic echinococcosis in other organs.Single Echinococcus granulosus infection is very rare.CASE SUMMARY In this report,we presented a case of primary pelvic Echinococcus granulosus infection admitted to the First Affiliated Hospital of Xinjiang Medical University.We described the key diagnostic points and surgical treatment of this case.We also summarized the epidemiological characteristics and pathogenesis of the disease.CONCLUSION Our case may provide clinical data for the diagnosis and treatment of primary pelvic Echinococcus granulosus infection.

Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.

Echinococcus Granulosus 14-3-3 Protein:A Potential Vaccine Candidate Against Challenge with Echinococcus Granulosus in Mice

Objective To investigate the protective immunity against Echinococcus granulosus in mice immunized with rEg14-3-3. Methods ICR mice were subcutaneously immunized three times with rEg14-3-3, followed by the challenge with Echinococcus granulosus protoscoleces intraperitoneally and then sacrificed after six months of post-challenge to detect the proliferation of splenocytes by MTT assay, and to measure the secretion of IL-2, IL-4, IL-20, and IFN -y by ELISA. The rate of reduced hydatid cyst and the levels of IgE, igG and IgG subclasses in sera were examined. Results Mice vaccinated with rEg14-3-3 and challenged with protoscoleces revealed significant protective immunity of 84.47%. ELISA analysis indicated that the immunized mice generated specific high levels of IgG and the prevailing isotypes of IgG were IgG1 and IgG2a. Splenocytes from mice immunized with rEg14-3-3 showed a significant proliferation response. The secretion of IFN-V and IL-2 increased significantly in the vaccinated mice whereas there was no significant difference in IL-4 and IL-20 levels between vaccinated and control mice. Conclusion The results indicate that the rEg24-3-3 vaccine could induce a high level of protective immunity as a promising vaccine candidate to prevent cystic echinococcosis.

Analysis of Protoscoleces-specific Antigens from Echinococcus Granulosus with Proteomics Combined with Western Blot

Objective To establish and optimize the proteomic analysis of protoscoleces-specific antigens from Echinococcus granulosus. To provide a foundation for identifying specific antigens in the soluble proteins of E. granulosus protoscoleces for further research. Methods Brood capsules were collected aseptically from fertile E. granulosus cysts from the livers of an infected patient. The fertile E. granulosus cysts were fractured, and protoscoleces were collected by centrifugation. The soluble proteins of protoscoleces were acquired using the 2D Quant kit according to the manufacturer＇s instructions. We employed two-dimensional electrophoresis （2-DE） combined with immunoblot assay （Western blot） to analyze the soluble components of E. granulosus protoscoleces antigens. The 2-DE and immunoblot maps obtained were analyzed with PDQuest 8.0 image analysis software. Results About 233 soluble protein spots were identified with Coomassie-stained gels. Most of the proteins had a molecular weight of 16 000 Da to 117 000 Da, and an isoelectric point value of 3.0 to 10.0. 2-DE immunoblot was conducted and 57 specific antigen spots were observed, among which 23 spots were identified. Conclusion 2-DE combined with Western blot is the key to successful proteomic analysis and presents a new possibility for searching the specific E. granulosus protoscoleces antigens.

Genetic diversity and phylogenetic analysis of EG95 sequences of Echinococcus granulosus:Implications for EG95 vaccine application

Objective:To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus(E. granulosus). Methods:We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1(DNASTAR Inc.,Madison,WI),and the phylogenetic analysis was performed by using MEGA5.1(CEMI,Tempe,AZ,USA). In addition,linear and conformational epitopes were analysed,including secondary structure,NXT/S glycosylation,fibronectin type ecoⅢ(Fnndary Ⅲ) domain and glycosylphosphatidylinositol anchor signal(GPIanchor). The s structure was predicted by PSIPRED method. Results:Our results indicated that most isolates overall shared 72.6-100% identity in EG95 gene sequence with the published standard EG95 sequence,X90928. However,EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates,respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine,X90928,and they belonged to Subgroup 1. However,in comparison to X90928,several amino acid mutations occurred in most isolates besides oncosphere,which potentially altered the immunodominant linear epitopes,glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine. Conclusions:This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates,and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

In-vitro scolicidal activity of Mallotus philippinensis (Lam.) Muell Arg. fruit glandular hair extract against hydatid cyst Echinococcus granulosus

Objective: To investigate new scolicidal agent from natural resources to cope with the side effects associated with synthetic drugs in Echinococcosis. Methods: The scolicidal potential of methanolic fruit powder extract (10 and 20 mg/mL) of Mallotus philippinensis ( M. philippinensis ) was investigated. Viability of protoscoleces was confirmed by trypan blue exclusion method, where mortality was observed at concentration of 10 and 20 mg/mL in 60 min treatment against Echinococcus granulosus ( E. granulosus ), under in-vitro conditions with reference to the known standard drug Praziquantel . Results: At concentration 10 and 20 mg/mL, the mortality rate was observed 97% and 99% respectively for 60 min treatment; while up to 93% mortality was observed with 20 mg/mL for only 10 min treatment. The concentration above 20 mg/mL for above 2 h showed 100% mortality, irrespective of further incubation. Conclusions: As compared with the standard anti-parasitic drug Praziquantel our extract has significant scolicidal activity with almost no associated side effects.

Identification and bioinformatics analysis of lactate dehydrogenase genes from Echinococcus granulosus

Objective:To identify full length cDNA sequence of lactate dehydrogenase(LDH) from adult Echinococcus granulosus(E.granulosus) and to predict the structure and function of its encoding protein using bioinformatics methods.Methods:With the help of NCBI,EMBI, Expasy and other online sites,the open reading frame(ORF),conserved domain,physical and chemical parameters,signal peptide,epitope,topological structures of the protein sequences were predicted and a homology tertiary structure model was created:Vector NT1 software was used for sequence alignment,phylogenetic tree construction and tertiary structure prediction. Results:The target sequence was 1 233 bp length with a 996 bp biggest ORF encoding 331 amino acids protein with typical L-LDH conserved domain.It was confirmed as full length cDNA of LDH from E.granulosus and named as EgLDH(GenBank accession number:HM748917).The predicted molecular weight and isoelectric point of the deduced protein were 3 5516.2Da and 6.32 respectively.Compared with LDHs from Taenia solium,Taenia saginata asiatica,Spirometra erinaceieuropaei.Schistosoma japonicum,Clonorchis sinensis and human,it showed similarity of 86% ,85% ,55% ,58% ,58% and 53% ,respectively.EgLDH contained 3 putative transmembrane regions and 4 major epitopes(54aa-59aa.81aa-87aa,97aa-102aa,307aa-313aa),the latter were significant different from the corresponding regions of human LDH.In addition,some NAD and substrate binding sites located on epitopes 54aa-59aa and 97aa-102aa,respectively.Tertiary structure prediction showed that 3 key catalytic residues 105R,165D and 192H forming a catalytic center near the epitope 97aa-102aa,most NAD and substrate binding sites located around the center.Conclusions:The full length cDNA sequences of EgLDH were identified.It encoded a putative transmembrane protein which might be an ideal target molecule for vaccine and drugs.

Echinococcus Granulosus: Suitable in vitro Protoscolices Culture Density

The present study is to determine the suitable protoscolices （PSCs） density for long-time culturing in vitro. The PSCs were divided into eight groups with different densities and the viability tests were carried out with 0.1% methylene blue staining. Then the infection ability of cultured PSCs was assessed by the mean cyst weight of mice inoculated intraperitoneally with PSCs after 8 months post-infection.

Morphometric differentiation between camel and sheep strains of Echinococcus granulosus using computer image analysis system(CIAS)

Objective:To find importance of morphometrie criterion of larval rostellar hook of Echinococcus granulosus(E.granulosus) and the easy and reliable method for distinguish sheep and camel strains in epidemiologic studies.Methods:Larval rostellar hooks(n=1860) of 31 camel and sheep isolates in Iran,which aheady had been characterized by PCR.were carefully processed by computerized imagime analysis system(CIAS) and acquired data about rostellar books were analyzed using software SPSS.Results:Measurement analysis of rostellar hooks[mean Iength (24.23±3.12lμm]indicated that length of the large hook was a remarkable parameter for strain differentiation.Data analysis demonstrated that CIAS could be used as a reliable tool to distinguish camel from sheep strains with high sensitivity(95.2%) and specificity(91.5%). Conclusions:CIAS as a specific,sensitive,economic,fast,and reliable means might be used for differentiation of E.granulosus strains.Although perimeter and area were measured by digital technology,they were not shown as discriminative criterion as total hook length did.

Study of Infection of Echinococcus granulosus in Yak in Spring and Its Potential Role in Transmission of Cystic Echinococcosis in Rangtang County of Sichuan,China

Dear Editor, Cystic echinococcosis （CE）, caused by Echinococcus granulosus in larval stage, is considered as one of the most dangerous parasitic zoonosis in the world. The obligate 2-host parasitic cycle of Echinococcus granulosus is predominantly synanthropic. Dogs are the usual definitive hosts, and lots of mammalian species can be intermediate hosts, including domestic livestock and human[I2]. In the Tibetan plateau, China, the population is mainly Tibetans primarily engaged in livestock husbandry and CE is therefore a health problem for both people and animal in Tibetan communities. The reported infection rate of Echinococcus gronulosus in slaughtered yak in slaughterhouses is usually very high, being about 50% or higher as reported, and the liver and lungs are the main affected organs[34].

Molecular differentiation of cryptic stage of Echinococcus granulosus and Taenia species from faecal and environmental samples

Objective:To differentiate cryptic stage of Echinococcus granulosus(E.granulosus) and Taenia by PCR-RFLP and sequence information of amplicon.Methods:DNA were isolated from metacestodes stage of Taenia and E.granulosus using DNA isolation kit(Q-BIOgene kit,USA), the amplified and purified DNA product was then cloned and sent for sequencing.The generating sequence information was used for amplicons identification.Results:Out of 112 faecal and environmental samples,16 exhibited positive result.The product size of amplicon positive for E.granulosus was 310 bp;whereas,for Taenia spp.sizes varied from 379 to 388 bp.Restriction pr of ile of actinⅡwith Csp61 also differed Taenia spp.and E.granulosus.Conclusions:The result of the study indicated that,the primers were useful to differentiate cryptic stage of the two genera which is yet to be reported earlier.

Molecular characterization of Echinococcus granulosus in paraffin-embedded human tissues from Southwest Iran

Objective:To investigate Echinococcus(E.)granulosus genotypes as the causative agents of hydatidosis in humans in the southwest of Iran(Khuzestan province).Methods:In this study,isolates of 80 archived human paraffin embedded hydatid cysts were collected from pathology laboratories in Ahvaz city,Khuzestan province.DNA was extracted and examined by nested-PCR of ribosomal DNA(rDNA)internal transcribed spacer 1(ITS1),and PCR-RFLP.In addition,the sequences of fragments of genes coding for Cox space1 and NADH dehydrogenase 1(ND1)were also examined.Results:Of the 80 paraffin samples,44(55.0%)were from the liver,27(33.8%)from the lung,and the rest from other organs.The amplified hydatid genomic DNA showed that the cysts were E.granulosus strains.The results of PCR-RFLP and sequencing analysis revealed the presence of G1 genotype(sheep strain)in all human isolates.Furthermore,no camel strain(G6)was detected among all samples in the regions studied.Conclusions:The molecular findings indicate that the predominant genotype involved in E.granulosus transmission in southwest of Iran is the common sheep strain(G1),which occurs in human populations.These results may have important implications for hydatid disease control in the studied areas.

In vitro treatments of Echinococcus granulosus with fungal chitosan,as a novel biomolecule

Objective:To determined the antiparasitic activity of the isolated chitosan from Penicillium viridicatum,Penicillium aurantiogriseum and commercial chitosan against protoscolicidal of hydatid cysts were determined.Methods:After isolating chitosan from fungal cell walls,four concentrations(50,100,200,400μg/mL)of each type of prepared chitosan and commercial chitosan were used for 10,30,60,and 180min,respectively.Results:Among different type of chitosan,commercial chitosan with the highest degree of deacetylation showed high scolicidal activity in vitro.Fungal chitosan could be recommended,as good as commercial chitosan,for hydatic cysts control.Conclusions:It seems to be a good alternative to synthetic and chemical scolicidal.

Echinococcus granulosus:A novel parenchymal sparing surgical treatment

Human echinococcosis is a zoonosis caused by Echinococcus(E.)tapeworms.There are different species,but E.granulosus and E.multilocularis have clinical relevance causing cystic and alveolar echinococcosis,respectively.Cystic echinococcosis(CE)is defined as presence of one or more hydatid cysts,most frequently in the liver and the lungs,less usual in the kidneys,bones,spleen,pancreas,heart or brain.The location,the number and size of the cysts determine the severity of the disease[1].

Molecular Characterization of <i>Echinococcus granulosus</i>in South of Iran

The focus of present study was to determine the epidemiological and molecular aspects of different strains of cystic echinococcosis in Fars province, Iran. Liver and lung samples from 410 sheep, 206 goats and 315 cattle were collected. In cattle, the infestation rate was 18.1% (57/315), with 11.1% hepatic cysts and 7.0% pulmonary cysts. Out of all identified cysts, 31.4% of the hepatic and 31.8% of the pulmonary cysts were found fertile. Incidence rate of hydatid cyst infection in sheep was 15.5% (64/410) with 11.9% hepatic cysts and 3.6% pulmonary cysts, of which 24.5% and 20% of hepatic and pulmonary cysts were respectively identified as fertile. The infestation rate was 16.0% (33/206) in goat, in which 10.2% and 5.8% cysts were collected from liver and lung, correspondingly. The prevalence of fertile hepatic and pulmonary cysts was recorded as 23.8% and 16.7%, respectively. Genotyping the cystic materials using PCR showed that the most prominent strains responsible for cystic echinococcosis in the Fars province are G1 and G6/7, while no evidence of E. multilocularis was recorded. This information may give us some clues to find out more about strains distribution in different regions in Iran, which may finally use to find tools in the eradication program of the disease, here and elsewhere.

A COMPREHENSIVE ECHINOCOCCUS PREVENTION LAUNCHED IN THE TIBET AUTONOMOUS REGION

Echinococcus has been shared by both humans and animals for quite a long time now.The Tibet Autonomous Region（TAR）is one of the areas with severe epidemic of echinococcus,with patients and their families encountering devastating health problems and heavy economic burdens in relation to the disease,not to mention a dramatic loss of agriculture and animal husbandry production.In March 2017,

Immunology of a unique biological structure:the Echinococcus laminated layer

The larval stages of the cestode parasites belonging to the genus Echinococcus grow within internal organs of humans and a range of animal species.The resulting diseases,collectively termed echinococcoses,include major neglected tropical diseases of humans and livestock.Echinococcus larvae are outwardly protected by the laminated layer(LL),an acellular structure that is unique to this genus.The LL is based on a fibrillar meshwork made up of mucins,which are decorated by galactose-rich O-glycans.In addition,in the species cluster termed E.granulosus sensu lato,the LL features nano-deposits of the calcium salt of myo-inositol hexakisphosphate(Insp_(6)).The main purpose of our article is to update the immunobiology of the LL.Major recent advances in this area are(i)the demonstration of LL“debris”at the infection site and draining lymph nodes,(ii)the characterization of the decoy activity of calcium Inp6 with respect to complement,(iii)the evidence that the LL mucin carbohydrates interact specifically with a lectin receptor expressed in Kupffer cells(Clec4F),and(iv)the characterization of what appear to be receptor-independent effects of LL particles on dendritic cells and macrophages.Much information is missing on the immunology of this intriguing structure:we discuss gaps in knowledge and propose possible avenues for research.

Infection of sheep by Echinococcus multilocularis in Gansu,China:evidence from mitochondrial and nuclear DNA analysis

BackgroundIn the normal life cycle of the parasite(Echinococcus multilocularis)that causes alveolar echinococcosis,domestic and wild carnivores act as definitive hosts,and rodents act as intermediate hosts.The presented study contributes to the research on the distribution and transmission pattern of E.multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province,China.MethodsFrom 2020 to 2021,nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination.A near complete mitochondrial(mt)genome and selected nuclear genes were amplified from the cyst-like lesion for identification.To confirm the status of the specimen,comparative analysis with reference sequences,phylogenetic analysis,and network analysis were performed.ResultsThe isolates displayed≥98.87%similarity to E.multilocularis NADH dehydrogenase sub-unit 1(nad1)(894 bp)reference sequences deposited in GenBank.Furthermore,amplification of the nad4 and nad2 genes also confirmed all nine samples as E.multilocularis with>99.30%similarity.Additionally,three nuclear genes,pepck(1545 bp),elp-exons VII and VIII(566 bp),and elp-exon IX(256 bp),were successfully amplified and sequenced for one of the isolates with 98.42%similarity,confirming the isolates were correctly identified as E.multilocularis.Network analysis also correctly placed the isolates with other E.multilocularis.ConclusionsAs a result of the discovery of E.multilocularis in an unusual intermediate host,which is considered to have the highest zoonotic potential,the result clearly demonstrated the necessity for expanded surveillance in the area.

A multiplex PCR for differential detection of Echinococcus granulosus sensu stricto,Echinococcus multilocularis and Echinococcus canadensis in China

Background:Echinococcosis caused byEchinococcus is one of the most major infectious diseases in north-west highland of China.E.granulosus sensu strict,E.multilocularis,andE.canadensis are known to be the only three species related to human health transmitting in the areas.To achieve targeted treatment and control of echinococcosis,the accurate identification and discrimination of the species are important.However,currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency.Methods:In the study,a set of primers were designed to aim at the three human-pathogenicEchinococcus species in China.The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity.A total of 73parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method.Results:The multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products.The detection limit of the assay for each of the three species was 5 pg/μl when they were tested separately.When DNA mixtures of the targeted species containing the same concentration were used as templates,the lowest amount of DNA which can be detected was 50 pg/μl,10 pg/μl and 5 pg/μl forE.granulosus s.s.,E.multilocularis,andE.canadensis respectively.No cross-reactivity was observed when DNA from eight genetically close species was used as control templates.The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected withE.shiquicus,which showed negative signals in the developed assay.Of all the tested stool materials,16 were previously found positive forEchinococcus by visual and microscopic examination.Among these 16 samples,13 were confirmed by the multiplex PCR,and the other three tested negative.Additionally,the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms.Conclusions:The developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation,which proven to be a promising tool utilized in clinic and surveillance system.

Complementary comments on metastatic liver lesions with exceptional and rare cases

Liver metastases can appear in different forms in magnetic resonance imaging.Contrary to popular belief,while radiologists report hypovascular or hypervascular metastatic lesions,exceptional examples may be detected in various tumors.The aim of this article is to improve this review by presenting rare and atypical examples of liver metastasis,as well as cases that might potentially be misdiagnosed as metastases during the process of differential diagnosis.