Immunohistochemistry as a Reliable Tool for the Diagnosis of Cystic Echinococcosis in Patients from Sardinia, Italy-A Confirmatory Study.

Cystic Echinococcosis (CE) is a zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s.l.). This study aims to investigate the use of two monoclonal antibodies (mAbEmG3 and mAbEm2G11) by immunohistochemistry (IHC) to confirm the diagnosis of CE in human patients, in particular in those cases in which other techniques fail to provide a correct or conclusive diagnosis. For this purpose, a survey on 13 patients was performed. These subjects were referred to Sardinian hospitals (Italy) from 2017 to 2022 and were suspected to be affected by CE. Our findings from these 13 patients showed the detection of E. granulosus sensu stricto by IHC in 12 of 13 echinococcal cysts, as one sample was of a non-parasitological origin. The results confirmed that IHC, by means of the mAbEmG3 and mAbEm2G11, is a reliable diagnostic tool that showed a very high performances when tested on strain of E. granulosus s.l. from Sardinia.

Molecular phylogenetic analysis of Echinococcus granulosus sensu lato infecting sheep in Italy.

Cystic Echinococcosis (CE), caused by the larval form of Echinococcus granulosus sensu lato, is a neglected zoonosis still threatening public health worldwide. In Italy different epidemiological scenarios were reported depending on the geographical area and associated socio-economic activities. Although in northern Italy the occurrence of E. granulosus is considered sporadic, in the southern regions and, particularly in Sardinia, CE prevalence reaches high levels. We analysed CE cysts collected from infected sheep from various areas of mainland Italy and the Sardinia island, with the main objective to investigate intergenotypic and intragenotypic variations at national level. CE cysts were collected from slaughtered sheep following post mortem inspection at local abattoirs. Total genomic DNA was extracted and amplification and sequencing of the partial mitochondrial genes nad5 and cox1 were performed. A Bayesian phylogenetic tree was estimated on a nad5 dataset (n = 260) composed of E. granulosus samples from this study (n = 126) and all the nad5 haplotypes available in GenBank (n = 134). In addition, haplotype network, diversity and neutrality analysis were performed on nad5 and cox1 sequences of Italian origin obtained in this study. E. granulosus sensu stricto (s.s.) was found to be the only Echinococcus species infecting sheep in Italy, mainly represented by G1 genotype (76 %) and, to a lower extent, by G3 genotype (24 %). Phylogenetic analyses revealed 40 nad5 and 33 cox 1 haplotypes, and the presence of two founder haplotypes, belonging to G1 and G3 genotype, showing 100 % similarity with DNA sequences from different geographic regions. The lack of geographical segregation, high haplotype and low nucleotide diversity coupled with significant negative values of Tajima's D and Fu's Fs observed in this study indicated high genetic variation and demographic expansion of E. granulosus s.s. in Italy.

Genetic Characterization of Echinococcus granulosus sensu stricto Isolated from Human Cysts from Sardinia, Italy.

This study involved 20 patients affected by cystic echinococcosis (CE) who were referred to different hospitals of Sardinia (Italy) from 2017 to 2022. By means of a multidisciplinary approach, diagnosis was confirmed for CE in 18 patients and for different aetiologies in two subjects. Moreover, serology was positive for 15 subjects. Since multiple CE cysts were found in five patients, a total of 27 lesions were collected; however, only one for each patient was investigated for genetic characterization of E. granulosus s.s. DNA isolates. Our results included 15 fertile cysts that underwent DNA extraction and amplification by three different PCRs targeting nuclear (calreticulin) and mitochondrial genes (cox1 and nad5). DNA was sequenced, and by neighbour-joining phylogenetic trees we determined 10 G1 and five G3 genotypes previously reported in Sardinia. These sequences were used to construct a network, along with those circulating in Mediterranean areas. The haplotype network calculated on cox1 evidenced seven different haplotypes of the 15 isolates, with SAR2 the most represented, carried by seven cysts, and SAR17 never described in the Mediterranean area. Meanwhile, the nad5 sequences showed the most common haplotype as nd5SAR7, as well as two new haplotypes not previously described, nd5SAR13, isolated from a Sardinian patient, and nd5SAR14, isolated from a Romanian patient.

Detection of Echinococcus granulosus sensu lato in Environmental Samples from Ibadan, Oyo State, South West Nigeria.

Environmental contamination with parasite eggs poses a serious risk to public health. This study aimed to assess the presence of taeniid eggs and, in particular, E. granulosus s.l., in environmental samples in the city of Ibadan, South West Nigeria. To this purpose, soil (n = 200), fecal (n = 200) and water samples (n = 50) were examined by microscopic observation and the multiplex PCR method. The influence of specific environmental factors on E. granulosus s.l. egg dispersion was also evaluated. Taeniid eggs were microscopically found in 11.5%, 25.5% and 8.0% of soil, fecal and water samples, respectively. PCR analyses evidenced the presence of E. granulosus s.l. in 8.0%, 24.0% and 2.0% of soil, fecal and water samples, respectively. The proximity to slaughterhouses, the level of urbanisation and the local government area of belonging did not seem to affect E. granulosus s.l. egg dissemination patterns. Our results have clearly demonstrated that both urban and semi-urban areas of the city of Ibadan in Nigeria are highly contaminated by taeniid eggs and we recommend the adoption of appropriate measures to control E. granulosus s.l.

Comparison and evaluation of analytic and diagnostic performances of four commercial kits for the detection of antibodies against Echinococcus granulosus and multilocularis in human sera.

Cystic echinococcosis (CE) is a disease caused by Echinococcus granulosus sensu lato (s.l.), an ubiquitous worldwide zoonotic agent affecting humans and animals. Diagnosis of CE in humans is usually performed by imagine techniques along with immunoassays. The aim of our study was to evaluate and compare four commercial diagnostic kits, based on the detection of IgG antibodies against E. granulosus and E. multilocularis. The study was performed on a total of 259 sera: the positive (n = 74) and the negative (n = 185) group. The following analytic and diagnostic performances of the four kits were evaluated: operator skills, specificity, sensitivity, repeatability, reproducibility, accuracy, positive and negative predictive values. Based on the parameters evaluated, all four tests demonstrated excellent quality and proved to be reliable diagnostic tools to support the clinical evaluation of human patients suspected of having CE. The four commercial assays, in our hands, presented altogether, a range of performances from good to excellent, being immunoblotting (IB) the most reliable, used as gold standard, followed by the immunochromatographic test (ICT) and finally the two enzyme linked immunosorbent assay (ELISAs).

Proteomic characterization of Echinococcus granulosus sensu stricto, Taenia hydatigena and Taenia multiceps metacestode cyst fluids.

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.

Prevalence of Echinococcus granulosus sensu lato in Owned Dogs in Lagos State, Nigeria.

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.

Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays.

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: Clinical, Immunological, and Biomolecular Evaluation of Patients from Sardinia (Italy).

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'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.

Bayesian Analysis of Three Methods for Diagnosis of Cystic Echinococcosis in Sheep.

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 (96.7-99.8), Sp of 97.5 (90.3-99.8), PPV of 97.6 (90.5-100), and NPV of 99.7 (96.5-100). The experimental design used in this work could be implemented as a validation protocol in a quality assurance system.

Genetic diversity of Echinococcus granulosus sensu stricto in Sardinia (Italy).

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.

Validation of a one-step PCR assay for the molecular identification of Echinococcus granulosus sensu stricto G1-G3 genotype.

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.

Cystic echinococcosis in a domestic cat (Felis catus) in Italy.

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.

Level of neurotoxic metals in amyotrophic lateral sclerosis: A population-based case-control study.

The association between exposure to toxic metals and amyotrophic lateral sclerosis (ALS) was explored in a population-based case-control study in the Sardinia island (Italy), a region characterized by elevated rates of ALS cases. In 34 patients with ALS (mean age, 62 ± 10 years) and 30 controls (mean age, 65 ± 11 years), Al, Cd, Hg, Mn and Pb were determined in blood, hair and urine by sector field inductively coupled mass spectrometry. Results indicated that, in blood, concentrations of Al (p=0.045) and Pb were higher (p=0.026) in ALS patients than in control subjects. In hair, a depletion of Al (p=0.006) and Mn (p=0.032) concentrations in ALS subjects respect to controls was found. In urine, no significant differences between cases and controls were observed. Thus, some metals seemed to be associated with ALS degeneration, but a definitive conclusion is still far considering the multiple risk factors (genetic mutations, environmental toxicants and stressors) involved in the disease. Finally, the interpretation that deregulated metal concentrations can be a consequence of the degenerative process, rather than a cause, is also valid.

Association of trace elements with lipid profiles and glycaemic control in patients with type 1 diabetes mellitus in northern Sardinia, Italy: An observational study.

Sardinia is an Italian region with a high incidence of type 1 diabetes mellitus. This study aimed to determine the associations of trace elements with lipid profiles and glycaemic control in patients with T1DM. A total of 192 patients with T1DM who attended the Unit of Diabetology and Metabolic Diseases in Sassari, Italy, were enrolled. Trace elements zinc, copper, selenium, chromium, and iron were measured in whole blood by sector field inductively coupled plasma mass spectrometry. The correlations between metabolic variables and the levels of trace elements were determined. Zinc was positively correlated with total cholesterol (P=0.023), low-density lipoprotein (P=0.0015), and triglycerides (P=0.027). Iron as significantly correlated with TC (P=0.0189), LDL (P=0.0121), and high-density lipoprotein (HDL) (P=0.0466). In males, Cr was positively correlated with HDL (P=0.0079) and Se, in females was correlated with TG (P=0.0113). The mean fasting plasma glucose was166.2mgdL(-1). Chromium was correlated with fasting plasma glucose (P=0.0149), particularly in males (P=0.0038). Overall, 63.5% of the patients had moderate HbA1c (7-9%). Copper was significantly correlated with HbA1c% in males (P=0.0155). In conclusion, the results of this study indicate that trace elements show different associations with lipid levels and glycaemic control in T1DM. Zinc, Fe, and Se were associated with lipid levels whereas Cu and Cr were associated with HbA1c%.

Blood metals concentration in type 1 and type 2 diabetics.

Mechanisms for the onset of diabetes and the development of diabetic complications remain under extensive investigations. One of these mechanisms is abnormal homeostasis of metals, as either deficiency or excess of metals, can contribute to certain diabetic outcomes. Therefore, this paper will report the blood levels of chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), mercury (Hg), nickel (Ni), lead (Pb), selenium (Se), and zinc (Zn) in subjects with type 1 diabetes (n = 192, mean age 48.8 years, mean disease duration 20.6 years), type 2 diabetes (n = 68, mean age 68.4 years, mean disease duration 10.2 years), and in control subjects (n = 59, mean age 57.2 years), and discuss the results indicating their possible role in diabetes. The metal concentrations were measured by sector field inductively coupled plasma mass spectrometry after microwave-induced acid digestion of blood samples. The accuracy was checked using a blood-based certified reference material, and recoveries of all elements were in the range of 92-101 % of certified values. Type 1 diabetes was found to be associated with Cr (p = 0.02), Mn (p < 0.001), Ni (p < 0.001), Pb (p = 0.02), and Zn (p < 0.001) deficiency, and type 2 diabetes with Cr (p = 0.014), Mn (p < 0.001), and Ni (p < 0.001) deficiency. These deficiencies were appreciated also subdividing the understudied patients for gender and age groups. Furthermore, in type 1 diabetes, there was a positive correlation between Pb and age (p < 0.001, ρ = 0.400) and Pb and BMI (p < 0.001, ρ = 0.309), while a negative correlation between Fe and age (p = 0.002, ρ = -0.218). In type 2 diabetes, there was a negative correlation between Fe and age (p = 0.017, ρ = -0.294) and Fe and BMI (p = 0.026, ρ = -0.301). Thus, these elements may play a role in both forms of diabetes and combined mineral supplementations could have beneficial effects.