Human alveolar echinococcosis in Slovakia: Epidemiology and genetic diversity of Echinococcus multilocularis, 2000-2023.

Human alveolar echinococcosis (AE) is a serious parasitic disease caused by larval stages of Echinococcus multilocularis. Between January 2000 and October 2023, 137 AE cases were confirmed in Slovakia. The average annual incidence increased from 0.031 per 100,000 inhabitants between 2000 and 2011, to an average of 0.187 since 2012, i.e. about six times. Among patients, 45.3% were men and 54.7% were women; the mean age at the time of diagnosis was 52.8 years. Most cases were diagnosed in the age groups 51-60 years and 61-70 years (33 cases each), and eight patients fell into the age category ≤ 20 years. To better recognize the gene diversity in clinical samples, metacestodes from 21 patients collected between 2013 and 2021 were subjected to DNA sequencing of four mitochondrial genes. Using concatenated sequences of cob (603 bp), nad2 (882 bp) and cox1 (789 bp) gene fragments, 14 isolates (66.7%) were assigned to the European E5 profile of E. multilocularis, two isolates (9.5%) to the E5a subtype, four isolates (19%) to the E4 profile, and one isolate (4.8%) to haplogroup E1/E2. The E5-type profiles and E4 profiles were distributed throughout the country, whereas the E1/E2 profile was found in the patient from western Slovakia. According to the data obtained and GenBank sequences, the E5-type dispersal is so far limited to central-eastern Europe and the variant seems to be indigenous to that region. The admixture with the haplotypes E4 and E1/E2 could have taken place from a historical endemic focus during the fox expansion in the last decades. By employing the nad1 fragment, a typical European haplotype was observed in all 21 resolved Slovak samples. The acceleration in the AE incidence in the last decade suggests the emergence of the disease and the need for further research on human and animal isolates.

A Case of Gastroallergic and Intestinal Anisakiasis in Italy: Diagnosis Based on Double Endoscopy and Molecular Identification.

Nematodes of the genus Anisakis (Rhabditida, Anisakidae) are zoonotic fish-borne parasites and cause anisakiasis, a disease with mild to severe acute or chronic gastrointestinal and allergic symptoms and signs. Anisakiasis can potentially lead to misdiagnosis or delay in diagnosis, and it has been suggested as a risk factor for gastrointestinal tumors. Here, we describe a case report of a 25-year-old woman who presented with gastrointestinal (abdominal pain, nausea, diarrhea) and allergic (diffuse skin rash) symptoms and reported ingestion of raw fish contaminated by worms. Gastro and colon endoscopy allowed the visualization and removal of nematodes and collection of bioptic tissue from ulcers and polyps. The removed nematodes were molecularly identified as Anisakis pegreffii. The patient was treated with chlorphenamine maleate, betamethasone, omeprazole, paracetamol, albendazole. We conclude that an upper endoscopy matched with a colonoscopy and molecular characterization of the pathogen yields the most reliable diagnosis and treatment for human anisakiasis, enabling the complete removal of the larvae and preventing chronic inflammation and damage.

Prevalence and Molecular Characterisation of Blastocystis sp. Infecting Free-Ranging Primates in Colombia.

Infection with Blastocystis sp. has been reported in free-living and captive non-human primates (NHPs); however, surveys on Blastocystis sp. from north-western South America are scarce. This study aimed to identify Blastocystis sp. in free-ranging NHPs living in Colombia. A total of 212 faecal samples were collected from Ateles hybridus, Cebus versicolor, Alouatta seniculus, Aotus griseimembra, Sapajus apella, and Saimiri cassiquiarensis. Smears and flotation were used for morphological identification. For samples microscopically classified as positive for Blastocystis sp., we used conventional PCR to amplify and sequence two regions of the SSU rRNA gene and used Maximum Likelihood methods and Median Joining Network analyses for phylogenetic analyses. Via microscopy, 64 samples were Blastocystis sp. positive. Through molecular analyses, 18 sequences of Blastocystis sp. subtype 8 (ST8) were obtained. Strain and allele assignment together with a comparative phylogenetic approach confirmed that the sequences were ST8. Alleles 21, 156, and 157 were detected. Median Joining network analyses showed one highly frequent haplotype shared by specimens from Colombia and Peru and close relationships between haplotypes circulating in NHPs from Colombia, Ecuador, Brazil, and Mexico. This survey could support the elaboration of a more accurate epidemiological picture of the Blastocystis sp. infecting NHPs.

Identification of Contracaecum rudolphii (Nematoda: Anisakidae) in Great Cormorants Phalacrocorax carbo sinensis (Blumenbach, 1978) from Southern Italy.

In this study, four dead great cormorant Phalacrocorax carbo sinensis (Blumenbach, 1978) specimens, collected from the coasts and lakes of Southern Italy, were examined by necropsy for the detection of Contraceacum sp. The adults and larvae found were subjected to morphological analysis and molecular identification by PCR-RFLP. A total of 181 Contracaecum specimens were detected in all of the four great cormorants examined (prevalence = 100%), showing an intensity of infestation between nine and ninety-two. A co-infestation by adult and larval forms of Contracaecum rudolphii was found only in one of the great cormorants examined. Following molecular investigations, 48 specimens of C. rudolphii A and 38 specimens of C. rudolphii B were detected, revealing co-infestation solely for the great cormorant from Leporano Bay (Southern Italy). Our results showed an opposite ratio between C. rudolphii A and C. rudolphii B in Pantelleria and in Salso Lake (Southern Italy) compared to what was reported in the literature, probably due to migratory stopovers and the ecology of the infested fish species, confirming the role of Contracaecum nematodes as ecological tags of their hosts.

Cuterebra baeri Infecting Grey-Legged Night Monkeys (Aotus griseimembra) and Red Howler Monkeys (Alouatta seniculus) in Colombia.

Myiases are parasitic infections caused by the larval stages of some fly species. In American nonhuman primates (NHP), three bot fly species causing cutaneous myiasis have been reported: Cuterebra baeri, Cochliomyia hominivorax, and Dermatobia hominis. Studies on myiasis in NHP are scarce and mainly based on morphologic identification of larvae, while molecular approaches have been barely used. This study aimed to molecularly identify bot flies parasitizing two NHP species living sympatrically in central Colombia. Bot fly larvae were collected from two grey-legged night monkeys (Aotus griseimembra) and from a howler monkey (Alouatta seniculus). Larvae were measured and photographed for morphologic evaluation; subsequent molecular characterizations of a partial region of the cytochrome c oxidase subunit 1 mitochondrial gene were performed. Sequence analysis allowed the identification of all specimens as Cuterebra baeri, enabling confirmation of this species parasitizing Alouatta seniculus and Aotus griseimembra in Colombia.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Intestinal parasites infecting captive non-human primates in Italy.

Non-human primates (NHPs) living in captive conditions are susceptible to intestinal parasites that can contribute to mortality and morbidity, and cause zoonotic infections. Thus, parasite surveys on NHP populations under human care are relevant as part of the evaluation of NHPs welfare and in the zoonotic disease risk assessment, as well as in the exploration of parasite transmission pathways, according to the One-Health concept. This study aimed to identify intestinal parasites infecting NHPs living in two wildlife recovery centers and in a zoological garden, in Italy. Ninety-three fecal samples from Macaca tonkeana, Macaca fascicularis, Sapajus apella, Chlorocebus aethiops, Macaca fuscata, Macaca sylvanus, and Cebus capucinus were collected at Piano dell'Abatino Park (Lazio), and fecal smears and flotation were performed in order to identify parasites according to morphological keys. Additionally, one carcass of M. fuscata from the Bioparco Zoological Garden of Rome (Lazio) and one of M. fascicularis from the Center for the Recovery of Exotic and Maremma Wild Animals (Tuscany) were necropsied and intestinal adult nematodes were collected and characterized at morphological and molecular level, using the mitochondrial cox1 and rrnL markers. Protozoans (Entamoeba coli, Iodamoeba bütschlii, Dientamoeba fragilis-like, Giardia sp.), chromists (Balantidium/Buxtonella sp.) and nematodes (Capillaria sp., Trichuris sp., strongyliform larvae and Oesophagostomum sp.) were found through fecal smears and flotation. The collected adult nematodes from dead NHPs were morphologically identified as whipworms (genus Trichuris). Phylogenetic analyses grouped Trichuris specimens into the Trichuris trichiura complex of species, with specimens from M. fuscata clustering into a host-specific branch, and whipworms from M. fascicularis clustering within a clade formed by Trichuris infecting several primate species, including humans. The results here collected revealed the presence of potentially zoonotic parasites circulating in captive primates in Italy, providing useful information for the formulation of management and care plans for captive NHPs, and for the elaboration of safety measures for visitors and animal keepers.

Inflammatory Response in Caco-2 Cells Stimulated with Anisakis Messengers of Pathogenicity.

Background: Anisakis spp. third-stage larvae (L3) are the causative agents of human zoonosis called anisakiasis. The accidental ingestion of L3 can cause acute and chronic inflammation at the gastric, intestinal, or ectopic levels. Despite its relevance in public health, studies on pathogenetic mechanisms and parasite-human interplay are scarce. The aim of this study was to investigate the human inflammatory response to different Anisakis vehicles of pathogenicity. Methods: Human colorectal adenocarcinoma (Caco-2) cells were exposed to Anisakis L3 (the initial contact with the host), extracellular vesicles (EVs, Anisakis-host communication), and crude extract (CE, the larval dying). The protein quantity and gene expression of two pro-inflammatory cytokines (IL-6 and IL-8) were investigated using an ELISA test (6 h and 24 h) and a qReal-Time PCR (1 h, 6 h, and 24 h), respectively. Results: The L3 and EVs induced a downregulation in both the Il-6 and Il-8 gene expression and protein quantity. On the contrary, the CE stimulated IL-6 gene expression and its protein release, not affecting IL-8. Conclusions: The Caco-2 cells seemed to not react to the exposure to the L3 and EVs, suggesting a parasite's immunomodulating action to remain alive in an inhospitable niche. Conversely, the dying larva (CE) could induce strong activation of the immune strategy of the host that, in vivo, would lead to parasite expulsion, eosinophilia, and/or granuloma formation.

What Do In Vitro and In Vivo Models Tell Us about Anisakiasis? New Tools Still to Be Explored.

Anisakiasis is a zoonosis caused by the ingestion of raw or undercooked seafood infected with third-stage larvae (L3) of the marine nematode Anisakis. Based on L3 localization in human accidental hosts, gastric, intestinal or ectopic (extra-gastrointestinal) anisakiasis can occur, in association with mild to severe symptoms of an allergic nature. Given the increasing consumption of fish worldwide, the European Food Safety Authority declared Anisakis as an emerging pathogen. Despite its importance for public health and economy, the scientific literature is largely characterized by taxonomic, systematic and ecological studies, while investigations on clinical aspects, such as the inflammatory and immune response during anisakiasis, using a proper model that simulates the niche of infection are still very scarce. The aims of this review are to describe the clinical features of anisakiasis, to report the main evidence from the in vivo and in vitro studies carried out to date, highlighting limitations, and to propose future perspectives in the study field of anisakiasis.

Neglected Diseases-Parasitic Infections among Slovakian Children from Different Populations and Genotypes of Giardia duodenalis.

Children are most prone to parasitic infections. The objectives of the study were to examine the occurrence of parasitic infections in children from different populations and to perform molecular characterization of human Giardia duodenalis isolates. We examined 631 stool samples from Roma and non-Roma children for the presence of parasitic developmental stages. Samples were collected from three eastern Slovakia districts. The ages of the children ranged from 1 months to 17 years. Subsequently, the molecular characterization of human G. duodenalis isolates by PCR detected triosephosphate isomerase (tpi) and beta-giardin (bg) genes was performed. The overall prevalence of parasitic infection was 19.8%. Ascaris lumbricoides eggs were the most frequent, with an occurrence of about 13.8%. G. duodenalis cysts were present in 6.3% of samples. G. duodenalis isolates obtained from 13 children were subjected to DNA sequencing with tpi and bg genes. Five isolates were categorized as bearing subassemblage BIII, the three isolates as subassemblage BIV, one person was infected with a mixture of subassemblages BIII and BIV, four children had subassemblage AII, and one isolate revealed a structure corresponding with subassemblage AI. Our work is proof that poverty and poor hygiene contribute the most to public health problems associated with neglected parasitic diseases.

Rat and fish peripheral blood leukocytes respond distinctively to Anisakis pegreffii (Nematoda, Anisakidae) crude extract.

Infective third-stage larvae (L3) of the marine nematode Anisakis pegreffii cause inflammation and clinical symptoms in humans, their accidental host, that subside and self-resolve in a couple of weeks after L3 die. To characterise the differences in an early immune response of a marine vs. terrestrial host, we stimulated peripheral blood leukocytes (PBLs) of fish (paratenic host) and rat (accidental, human-model host) with A. pegreffii crude extract and analysed PBL transcriptomes 1 and 12 h post-stimulation. Fish and rat PBLs differentially expressed 712 and 493 transcripts, respectively, between 1 and 12 h post-stimulation (false discovery rate, FDR <0.001, logFC >2). While there was a difference in the highest upregulated transcripts between two time-points, the same Gene Ontologies, biological processes (intracellular signal transduction, DNA-dependent transcription, and DNA-regulated regulation of transcription), and molecular functions (ATP and metal ion binding) were enriched in the two hosts, showing an incrementing dynamic between 1 and 12 h. This suggests that the two distinct hosts employ qualitatively different transcript cascades only to achieve the same effect, at least during an early innate immunity response. Activation of later immunity elements and/or a combination of other host's intrinsic conditions may contribute to the death of L3 in the terrestrial host.

Molecular Study of Echinococcus granulosus Cestodes in Ukraine and the First Genetic Identification of Echinococcus granulosus Sensu Stricto (G1 Genotype) in the Country.

INTRODUCTION: Cystic echinococcosis is a globally distributed zoonotic disease of great medical and veterinary importance, which is caused by the cestode Echinococcus granulosus sensu lato. In Ukraine, two areas of the prominent circulation of the parasite are established, the southern steppe zone with sheep as the main transmitter, and the northern forest-steppe zone and Polissia, where pigs are mainly responsible for maintaining the E. granulosus transmission. METHODS: Given that only a few studies have so far addressed the genetic diversity of the parasite in Ukraine, we have sequenced partial mitochondrial genes of cytochrome c oxidase 1 (789 bp), NADH dehydrogenase 1 (602 bp) and 12S rRNA (333-334 bp) in pig metacestodes from the Sumy region (farms close to Sumy, northeastern Ukraine) and the Kyiv region (a farm in Bila Tserkva, central Ukraine). RESULTS: Four isolates from four pigs in the Sumy region were identified as E. canadensis (G7 genotype), the major E. granulosus s.l. species circulating in Eastern Europe, including the three microvariants (G7A, G7B, G7C). Three isolates from the two pigs in the Kyiv region were classified as E. granulosus s.s. (G1 genotype), including one microvariant (G1A). CONCLUSION: To our knowledge, this is the first genetic record of E. granulosus s.s. with the presumed highest infectivity and virulence among the E. granulosus s.l. species in Ukraine. The finding has implications for public health as local control programmes should take into consideration different development rate of this parasite in dogs and the greater risk of the species for human infection.

Parasites of Free-Ranging and Captive American Primates: A Systematic Review.

The diversity, spread, and evolution of parasites in non-human primates (NHPs) is a relevant issue for human public health as well as for NHPs conservation. Although previous reviews have recorded information on parasites in NHPs (Platyrrhines) in the Americas, the increasing number of recent studies has made these inventories far from complete. Here, we summarize information about parasites recently reported in Platyrrhines, attempting to build on earlier reviews and identify information gaps. A systematic literature search was conducted in PubMed, ISI Web of Science, and Latin American and Caribbean Health Sciences Literature (LILACS), and following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Ninety-three studies were included after the screening process. Records for 20 genera of NHPs, including 90 species were found. Most of the studies were conducted on captive individuals (54.1%), and morphological approaches were the most used for parasite identification. The most commonly collected biological samples were blood and stool, and Protozoa was the most frequent parasite group found. There is still scarce (if any) information on the parasites associated to several Platyrrhine species, especially for free-ranging populations. The use of molecular identification methods can provide important contributions to the field of NHPs parasitology in the near future. Finally, the identification of parasites in NHPs populations will continue to provide relevant information in the context of pervasive habitat loss and fragmentation that should influence both human public health and wildlife conservation strategies.

Ascariasis in a 75-year-old man with small bowel volvulus: a case report.

BACKGROUND: Ascaris lumbricoides and Ascaris suum are the most common soil-transmitted helminths of humans and pigs, respectively. The zoonotic potential of A. suum has been a matter of debate for decades. This study was aimed to present a case of human ascariasis caused by A. suum in southern Italy. CASE PRESENTATION: A 75-year-old man presented to the department of surgery in Avellino (southern Italy) complaining of abdominal pain and vomiting. Physical examination revealed bloating and abdominal tenderness. A computed tomography scan showed air-fluid levels and small bowel distension. During exploratory laparotomy a small bowel volvulus with mesenteritis was evident and surprisingly an intraluminal worm was detected. The worm was removed with a small enterotomy and identified as an adult female of A. suum based on morphological and molecular analysis. Faecal examination revealed the presence of unfertilized Ascaris eggs with an intensity of 16 eggs per gram (EPG) of faeces. The patient was treated with mebendanzole 100 mg twice a day for 3 days. The post-operative course was regular with re-alimentation after 3 days and discharge after 12 days. CONCLUSIONS: This report shows as A. suum can function as a relevant agent of human zoonosis. Therefore, in patients with bowel obstruction with no evident aetiology a helminthic infestation should be considered for an accurate diagnosis, especially in patients living in rural areas.

Novel Omics Studies on Anisakid Nematodes.

Parasitic nematodes infecting humans and animals are widely distributed in marine and terrestrial environments, causing considerable morbidity and mortality globally [...].

Genotyping of Ascaris spp. infecting humans and pigs in Italy, Slovakia and Colombia.

BACKGROUND: The systematics and taxonomy of Ascaris lumbricoides and Ascaris suum, two of the world's most widespread nematodes, still represent a highly debated scientific issue. Two different transmission scenarios have been described according to endemicity: separated host-specific transmission cycles in endemic regions, and a single pool of infection shared by humans and pigs in non-endemic regions. The swine roundworm A. suum is now recognized as an important cause of human ascariasis also in endemic areas such as China, where cross-infections and hybridization have also been reported, as well as in non-endemic regions like Italy. This study aimed to investigate the molecular epidemiology of human and pig ascariasis in three countries representing different epidemiological scenarios: Italy as a non-endemic country, Colombia as an endemic country, and Slovakia as a non-endemic country, but with a poor socio-economic context linked to some focal populations of Roma settlements. MATERIALS AND METHODS: A total of 237 nematodes were analysed: 46 from Colombia (13 from humans, 33 from pigs), 114 from Slovakia (20 from humans, 94 from pigs) and 77 from Italy (17 from humans and 60 from pigs). Genotyping by PCR-RFLP of nuclear (ITS) and sequencing of mitochondrial (cox1) target regions were performed. ITS genotypes were used to estimate the Hardy-Weinberg (HW) equilibrium according to hosts and country of origin. The partial cox1 sequences were used to analyse genetic polymorphisms according to hosts and country of origin, as well as to infer the network of haplotypes, their evolutionary relationships and geographical distribution. RESULTS: 110 quality cox1 sequences were obtained. Haplotype network revealed three main groups corresponding to clade A, B and C. Clade C included most of the human cases from Italy, while those from Slovakia and Colombia were grouped in clade B. Ascaris from Italian and Colombian pigs showed HW equilibrium at the ITS marker, while disequilibrium was found in A. lumbricoides from Slovak pigs, which suggest a high unexpected amount of roundworms of human origin circulating also in pigs. CONCLUSIONS: This study updates and extends the current understanding of Ascaris species and genotypes circulating in different epidemiological scenarios, with particular attention to the inclusion of human-derived Ascaris in the phylogenetic cluster C. Despite the evidence of HW equilibrium in the ITS in pig-derived Italian samples, the amount of genetic variation seems to support the existence of two closely related species.

An unpleasant souvenir: Endoscopic finding of Trichuris trichiura (Nematoda: Trichuridae).

Whipworms are responsible for up to 500 million cases of trichuriasis worldwide, with higher endemicity in tropical and sub-tropical countries. In non-endemic countries, trichuriasis can be accidentally diagnosed upon colonoscopy, often in the presence of negative microscopy. Here, we describe an incidental diagnosis of trichuriasis in an HIV patient residing in a non-endemic area (i.e., Turin, Italy), six months after his return from Antigua. The species-level diagnosis was made thanks to PCR-based molecular identification of Trichuris sp. following optical microscopy detection. Overall, this case highlights the importance of improving parasitic diseases diagnosis through cutting-edge clinical and laboratory diagnostic tools alongside advanced training of specialists in the area of parasitology.