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The Italian National Reference Center for Echinococcosis (CeNRE, Sassari, Italy) set up a diagnostic protocol of "one-step-PCR" useful for the detection of E. granulosus sensu stricto (E.g.s.s.) and the identification of its genotype (G1-G3). The purpose of this work was to perform the validation of the "PCR E.g.s.s." method. The procedures were performed employing the criteria of the World Organization for Animal Health as well as of the Italian Accreditation Body (ACCREDIA) based on the Regulation UNI CEI EN ISO/IEC 17025. Positive DNA samples belonging to E. granulosus, E. ortleppi, E. multilocularis, E. canadensis species were used for the experiments. Analytical specificity evidenced primer pairs Cal (Calreticulin l gene of 1001 bp) with an specificity higher respect to Ef1 (Elongation-Factor 1 Alpha gene of 706 bp) and NAD (Dehydrogenase-subunit 1 gene of 219 bp). The analytical sensitivity presented the capability to detect a very low amount of parasite DNA corresponding to a concentration of 12.5 pg/µl; accuracy and precision related to the operator performance, along with repeatability and reproducibility, evidenced high concordance among results and demonstrated an excellent κ values of Cohen. According to the good performance related to the evaluated parameters, the method "PCR E.g.s.s." was suitable for the validation procedure, and consequently, to be undergone to the accreditation process. In conclusion, the results demonstrated an elevated robustness and reliable features of the "PCR E.g.s.s." able to perform a rapid diagnosis of E. granulosus in only "one step", hence, it is likely to avoid the sequencing step.

Echinococcus granulosus sensu lato (s.l.) infection in dogs poses risk of transmission to their owners and family members. We determined the prevalence and factors associated with E. granulosus s.l. infection among owned dogs presented at veterinary clinics or hospitals in Lagos State, Nigeria. Fecal samples from 217 dogs were screened for the presence of taeniid eggs using a sedimentation test in a cross sectional study. The taeniid eggs were identified at molecular level using a multiplex PCR. A structured questionnaire was used to obtain data on intrinsic and extrinsic factors from 133 dog owners. Out of the 217 dog fecal samples, 13 (6.0%) had taeniid eggs, of which 12 (92.3%) were identified as Echinococcus granulosus s.l. We found that Echinococcus granulosus infection is present among owned dogs in Lagos State with an overall prevalence of 5.5%. Location of the veterinary clinics or hospital and purpose for keeping dogs were significant factors associated with E. granulosus infection among owned dogs. Dogs living in suburban areas and kept for security purposes or guarding have higher probability of infection. Appropriate and regular treatment of dogs with praziquantel is highly recommended to reduce risk of E. granulosus transmission to humans.

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

Cystic echinococcosis (CE), a zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s.l.), is a worldwide public health problem. Echinococcus granulosus sensu stricto (s.s.), associated with G1 and G3 genotypes, is endemic with high prevalence in the Mediterranean basin. The parasite&rsquo, s life cycle comprises definitive hosts (canids) and intermediate hosts (ruminants) and can occasionally involve humans. The main aim of this research was to confirm the diagnosis of 13 patients suspected of CE who presented different complications and needed the surgical removal of the cysts. We also wanted to understand and clarify more the diagnosis of echinococcosis in humans. For this purpose, the patients first underwent cyst evaluation by ultrasound (US), immunological analysis, and then total pericystectomy, followed by parasitological, histopathological, and molecular biology examinations of the cysts. US stadiated one CE1, one CE2, eight CE3b, one CE4, and two CE5, immunology evidenced nine positives, histopathology confirmed 11 CE cysts, of which 8 fertile presenting protoscoleces were identified as E. granulosus s.s. by molecular biology, genotyped as three G1 and four G3 by neighbor-joining (NJ) phylogenetic tree. In conclusion, the results showed that 11 patients were affected by E. granulosus s.s. G1 orG3, and 2 cystic neoformations were of non-parasitic origin.

Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites.

Diagnosis of cystic echinococcosis (CE) in sheep is essentially based on necropsy findings. Clinical symptoms can be easily overlooked, while the use of immunological tests is still not recommended for an intra vitam diagnosis. This study assessed the performances of three post-mortem laboratory methods in the diagnosis of ovine CE. In the absence of a single and accurate test as a gold standard, the results of multiple analytical tests can be combined to estimate diagnostic performance based on a Bayesian statistical approach. For this purpose, livers (n = 77), and lungs (n = 79) were sampled from adult sheep and examined using gross pathology, histopathology and molecular analyses. Data from the three diagnostic methods were analyzed using a Bayesian latent class analysis model to evaluate their diagnostic accuracy in terms of sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV). The gross pathology examination revealed excellent diagnostic capabilities in diagnosing ovine CE with an Se of 99.7 (96.7&ndash, 99.8), Sp of 97.5 (90.3&ndash, 99.8), PPV of 97.6 (90.5&ndash, 100), and NPV of 99.7 (96.5&ndash, 100). The experimental design used in this work could be implemented as a validation protocol in a quality assurance system.

Cystic echinococcosis (CE) is a severe parasitic zoonosis caused by the metacestode of the tapeworm Echinococcus granulosus sensu lato (s.l.). The disease has a global distribution representing a significant public health concern. Based on mitochondrial DNA analysis E. granulosus s.l. has been subdivided into five species: E. granulosus sensu stricto (s.s.) (G1, G3 genotype), E. equinus (G4 genotype), E. ortleppi (G5 genotype), E. canadensis (G6-G8, G10 genotype) and E. felidis. E. granulosus s.s., and in particular G1, is the most widespread genotype and the major responsible of human CE cases worldwide. In Italy G1 genotype is higly represented with larger percentages in some hyperendemic areas such as Sardinia. Molecular studies represent a valuable tool to improve our understanding of the E. granulosus epidemiology and CE control strategies. In the present study we investigated genetic variability of E. granulosus s.s. in Sardinia. To this purpose 83 hydatid cysts were collected from different animal species including humans and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was partially sequenced (720 bp). Nucleotide sequences from Mediterranean basin were also analyzed for comparison. The phylogenetic network revealed 30 haplotypes grouped around a predominant isolate that had been already reported from other world regions. Haplotype diversity (0.8495 ± 0.0336) and nucleotide diversity (0.003305 ± 0.002014) were similar in Sardinia respect to other Mediterranean countries. Neutrality indices obtained by Tajima's D and Fu's Fs test were significantly negative (p ≤ .01) suggesting expansion of Sardinian population. Low Fixation indices (Fst), ranging from negative values (Algeria, Greece, Spain, other part of Italy) to 0.089 (Albania, France), indicated absence of genetic differentiation, and gene flow between Sardinia and other Mediterranean countries.

Echinococcus granulosus sensu lato is a zoonotic agent with a life cycle consisting of definitive hosts (dogs and wild carnivores), and intermediate hosts (usually ungulates). Other animals and humans may accidentally ingest eggs and contract cystic echinococcosis, acting as aberrant hosts. A 3-year-old neutered female cat was brought to a veterinary practice in Sassari (Italy) with abdominal distension. Ultrasound showed multiple intraperitoneal vesicles, which on laparotomy were found to be metacestodes of E. granulosus. Videos of the extraction of cysts are provided. Phylogenetic analysis based on a fragment of the cytochrome oxidase subunit 1 (cox1) mitochondrial gene identified the isolate as E. granulosus sensu stricto genotype G1, the most common genotype circulating in Europe and the Mediterranean basin. This is the first case report of cystic echinococcosis in domestic cats from Italy.