Diagnosis of Human Trematode Infections.

Digenetic trematodes form a major group of human parasites, affecting a large number of humans, especially in endemic foci. Over 100 species have been reported infecting humans, including blood, lung, liver and intestinal parasites. Traditionally, trematode infections have been diagnosed by parasitological methods based on the detection and the identification of eggs in different clinical samples. However, this is complicated due to the morphological similarity between eggs of different trematode species and other factors such as lack of sensitivity or ectopic locations of the parasites. Moreover, the problem is currently aggravated by migratory flows, international travel, international trade of foods and changes in alimentary habits. Although efforts have been made for the development of immunological and molecular techniques, the detection of eggs through parasitological techniques remains as the gold standard for the diagnosis of trematodiases. In the present chapter, we review the current status of knowledge on diagnostic techniques used when examining feces, urine, and sputum and also analyze the most relevant characteristics used to identify eggs with a quick key for the identification of eggs.

Echinostomes and Other Intestinal Trematode Infections.

Intestinal trematodes are among the most common types of parasitic worms. About 76 species belonging to 14 families have been recorded infecting humans. Infection commonly occurs when humans eat raw or undercooked foods that contain the infective metacercariae. These parasites are diverse in regard to their morphology, geographical distribution and life cycle, which make it difficult to study the parasitic diseases that they cause. Many of these intestinal trematodes have been considered as endemic parasites in the past. However, the geographical limits and the population at risk are currently expanding and changing in relation to factors such as growing international markets, improved transportation systems, new eating habits in developed countries and demographic changes. These factors make it necessary to better understand intestinal trematode infections. This chapter describes the main features of human intestinal trematodes in relation to their biology, epidemiology, host-parasite relationships, pathogenicity, clinical aspects, diagnosis, treatment and control.

Immunology and pathology of echinostomes and other intestinal trematodes.

Intestinal trematodes constitute a major group of helminths that parasitize humans and animals with relevant morbidity and mortality. Despite the importance of the intestinal trematodes in medical and veterinary sciences, immunology and pathology of these helminth infections have been neglected for years. Apart from the work focused on the members of the family Echnistomatidae, there are only very isolated and sporadic studies on the representatives of other families of digeneans, which makes a compilation of all these studies necessary. In the present review, the most salient literature on the immunology and pathology of intestinal trematodes in their definitive hosts in examined. Emphasis will be placed on members of the echinostomatidae family, since it is the group in which the most work has been carried out. However, we also review the information on selected species of the families Brachylaimidae, Diplostomidae, Gymnophallidae, and Heterophyidae. For most of these families, coverage is considered under the following headings: (i) Background; (ii) Pathology of the infection; (iii) Immunology of the infection; and (iv) Human infections.

Intestinal parasitic infections and their potential risk factors among prison inmates in Valencia, Spain.

BACKGROUND: Despite the fact that prison inmates are a population at higher risk than other groups of suffering from intestinal parasite infections in relation to their living conditions, information about these diseases in prison environments is still scarce. Herein, we analyze the status of intestinal parasite infections in a Spanish prison. METHODS: A cross-sectional study involving 528 inmates was conducted from April to June 2022 among inmates at Centro Penitenciario Picassent (Valencia, Spain). Stool specimens were examined using the direct wet mount technique, the formol-ether concentration technique, and the Ziehl-Neelsen staining method. We used STATA 16.1 for data analysis. We consider a p-value less than 0.05 significant at a 95% confidence level. RESULTS: Of the 528 inmates (471 men and 57 women; a mean age of 41.94 years) enrolled in the study, 83 (15.7%) were infected. Only six species of protozoa were detected. The gut potential microeukaryotic pathobiont Blastocystis sp. was the predominant parasite, accounting for 37 (44.6%) of the infections. Gut parasite amebas (6.6%) and pathobionts (5.3%) were more prevalent than flagellates (2.3%). The prevalence of infection with pathogenic species (8.9%) was similar to that of non-pathogenic species (8.7%). Infection among men (15.2%) was higher than in women (0.6%) (p < 0.0001). In multivariate analysis, the country of birth (AOR = 0.31, 95% CI = 0.18-0.52) and the time spent in prison (AOR = 1.83, 95% CI = 1.06-3.14) were statistically significant associated to intestinal parasite infections (p < 0.0001 and p = 0.028, respectively). CONCLUSION: This study found low levels of intestinal parasite infections in the CPP which could be indicative of the implementation of sanitary measures in prison environments in Spain. The less time spent in prison favor the risk of having infection while the Spanish nationality of inmates could reduce the risk of infection. The main recommendation would be to introduce routine parasitological tests upon foreigners entering prison.

Imported taeniasis in the context of colon hydrotherapy.

We report a case of imported taeniasis, under the modality of "visiting friends and relatives", in the context of colon hydrotherapy. This technique allows the detection of proglottids, diagnosed in this case as Taenia saginata based on the gravid proglottid that presented more than 13 uterine branches and showed active motility. Moreover, the patient did not consume pork for religious reasons. The treatment with paromomicin sulfate was effective. In this case, a trip to Ethiopia, together with the ingestion of raw beef, was the cause of parasitization. It is highly advisable to obtain detailed information from the patient on their background, especially their travel and dietary histories.

Changes in resident microbiota associated with mice susceptibility or resistance to the intestinal trematode Echinostoma caproni.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode with no tissue phases in the definitive host that has been extensively used as an experimental model to study the factors that determine resistance against intestinal helminths. In E. caproni infections in mice, interleukin-25 (IL-25) plays a critical role and it is required for the resistance to infection. However, little is known on the factors that determine its production. Primary E. caproni infection in mice is characterized by the development of chronic infections and elevated worm recovery, in relation to a local Th1 response with elevated production of interferon-γ. However, partial resistance against secondary E. caproni infections in ICR (Institute of Cancer Research) mice is developed after the chemotherapeutic cure of a primary infection and the innately produced IL-25 after pharmacological treatment. In this paper, we analyse the potential role of intestinal microbiota in the production of IL-25, and the subsequent resistance to infection. For this purpose, we analysed the production of IL-25 under conditions of experimental dysbiosis and also the changes in the resident microbiota in primary infections, pharmacological curation and secondary infections. The results obtained showed that resident microbiota play a major role in the production of IL-25 and the appearance of members of the phylum Verrucomicrobia as a consequence of the curation of the primary infection could be related to the partial resistance to secondary infection.

Neglected food-borne trematodiases: echinostomiasis and gastrodiscoidiasis.

In the present paper, we review two of the most neglected intestinal food-borne trematodiases: echinostomiasis, caused by members of the family Echinostomatidae, and gastrodiscoidiasis produced by the amphistome Gastrodiscoides hominis. Both parasitic infections are important intestinal food-borne diseases. Humans become infected after ingestion of raw or insufficiently cooked molluscs, fish, crustaceans, amphibians or aquatic vegetables. Thus, eating habits are essential to determine the distribution of these parasitic diseases and, traditionally, they have been considered as minor diseases confined to low-income areas, mainly in Asia. However, this scenario is changing and the population at risk are currently expanding in relation to factors such as new eating habits in developed countries, growing international markets, improved transportation systems and demographic changes. These aspects determine the necessity of a better understanding of these parasitic diseases. Herein, we review the main features of human echinostomiasis and gastrodiscoidiasis in relation to their biology, epidemiology, immunology, clinical aspects, diagnosis and treatment.

ENTEROPARASITES IN A POPULATION IN DEPRIVED CONDITIONS FROM PROVINCE OF VALENCIA (SPAIN).

The autonomous Valencian Community (Spain) has experienced intense population growth with economic difficulties or deprivation in basic goods, ranking as the sixth Spanish autonomous region with the highest percentage of a population at risk of poverty or social exclusion. The objective of this work is to assess the level of enteroparasites in a Valencian population that meets certain deprivation conditions. A coproparasitological analysis was carried out in 460 users (children and adult relatives) of the 3 Casa Caridad schools in the province of Valencia. The parasite prevalence reached 31.7%, with a higher frequency of pathogens/potentially pathogenic species (66.4%) than non-pathogenic species (33.5%), although symptoms only appeared in 10.3% of those with pathogens/potentially pathogenic species. Of those parasitized, the presence of pathogens/potentially pathogenic species reached 20.6% in individuals living in crowded conditions and 17.5% in those living with pets. Presenting pathogens/potentially pathogenic species infection increased almost fourfold in those of southern European origin. In family groups, infection occurred among all their members, showing interpersonal parasitic transmission linked to both material deprivation and a lack of health education. Improving epidemiological surveillance, health education, and hygiene/sanitation facilities is essential to reduce or eliminate parasitic transmission among those who suffer from certain deprivation conditions.

Identification and Molecular Characterization of Four New Blastocystis Subtypes Designated ST35-ST38.

Three recent studies of Blastocystis epidemiology in mammalian hosts identified four novel sequences that appeared to share B. lapemi as the most similar sequence. However, full-length ssu rRNA gene sequences were not available to confirm the validity of these new subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-length reference sequences for each of the new subtypes. Additionally, phylogenetic analyses and pairwise distance comparisons were performed to confirm the validity of each of these new subtypes. We propose that the novel sequences described in this study should be assigned the subtype designations ST35-ST38. The full-length reference sequences of ST35-ST38 will assist in accurate sequence descriptions in future studies of Blastocystis epidemiology and subtype diversity.

Interleukin-25-mediated resistance against intestinal trematodes does not depend on the generation of Th2 responses.

BACKGROUND: The cytokine interleukin-25 (IL-25) is recognized as the most relevant initiator of protective T helper 2 (Th2) responses in intestinal helminth infections. This cytokine induces resistance against several species of intestinal helminths, including the trematode Echinostoma caproni. E. caproni has been extensively used as an experimental model to study the factors determining resistance to intestinal infections. In the study reported here, we assessed the role of IL-25 in the generation of resistance in mice infected with E. caproni. METHODS: The factors that determine the production of IL-25 in mice experimentally infected with E. caproni were determined, as were the consequences of IL-25 production in terms of polarization of the immune response and resistance to infection. RESULTS: Our results show that the role of IL-25 in the polarization of the immune response differs between the primary and secondary immune responses. IL-25 is required for the development of a Th2 phenotype in primary E. caproni infections, but it can also promote the differentiation to Th2 memory cell subsets that enhance type-2 immunity in memory responses. However, the development of Th2 responses does not induce resistance to infection. The Th2 phenotype does not elicit resistance, and IL-25 is responsible for the resistance regardless of its type-2 cytokine activity and activation of signal transducer and activator of transcription (STAT6). Alternative activation of macrophages induced by IL-25 can be implicated in the resistance to infection. CONCLUSIONS: In contrast to primary infection, secondary infection elicits a type-2 immune response even in the absence of IL-25 expression. Despite the development of a type-2 response, mice are susceptible to secondary infection associated with the lack of IL-25. Resistance to infection is due to the production of IL-25, which acts autonomously from Th2 response in terms of parasite clearance.

Secreted cathepsin L-like peptidases are involved in the degradation of trapped antibodies on the surface of Echinostoma caproni.

Antibody trapping is a recently described strategy for immune evasion observed in the intestinal trematode Echinostoma caproni, which may aid to avoiding the host humoral response, thus facilitating parasite survival in the presence of high levels of local-specific antibodies. Parasite-derived peptidases carry out the degradation of trapped antibodies, being essential for this mechanism. Herein, we show that cathepsin-like cysteine endopeptidases are active in the excretory/secretory products (ESPs) of E. caproni and play an important role in the context of antibody trapping. Cysteine endopeptidase activity was detected in the ESPs of E. caproni adults. The affinity probe DCG-04 distinguished a cysteine peptidase band in ESPs, which was specifically recognized by an anti-cathepsin L heterologous antibody. The same antibody localized this protein in the gut and syncytial tegument of adult worms. Studies with cultured parasites showed that in vivo-bound antibodies are removed from the parasite surface in the absence of peptidase inhibitors, while addition of cathepsin L inhibitor prevented their degradation. These results indicate that cathepsin L-like peptidases are involved in the degradation of surface-trapped antibodies and suggest that cysteine peptidases are not only crucial for tissue-invading trematodes, but they can be equally relevant at the parasite-host interface in gut-dwelling flukes.

Intestinal Trematode Infections.

Intestinal trematodes are among the most common types of parasitic worms. About 76 species belonging to 14 families have been recorded infecting humans. Infection commonly occurs when humans eat raw or undercooked foods that contain the infective metacercariae. These parasites are diverse with regard to their morphology, geographical distribution, and life cycle, which make it difficult to study the parasitic diseases that they cause. Many of these intestinal trematodes have been considered as endemic parasites in the past. However, the geographical limits and the population at risk are currently expanding and changing in relation to factors such as growing international markets, improved transportation systems, new eating habits in developed countries and demographic changes. These factors make it necessary to better understand intestinal trematode infections. This chapter describes the main features of human intestinal trematodes in relation to their biology, epidemiology, host-parasite relationships, pathogenicity, clinical aspects, diagnosis, treatment, and control.

Diagnosis of Human Trematode Infections.

Digenetic trematodes form a major group of human parasites, affecting a large number of humans, especially in endemic foci. Over 100 species have been reported infecting humans, including blood, lung, liver, and intestinal parasites. Traditionally, trematode infections have been diagnosed by parasitological methods based on the detection and the identification of eggs in different clinical samples. However, this is complicated due to the morphological similarity between eggs of different trematode species and other factors such as lack of sensitivity or ectopic locations of the parasites. Moreover, the problem is currently aggravated by migratory flows, international travel, international trade of foods, and changes in alimentary habits. Although efforts have been made for the development of immunological and molecular techniques, the detection of eggs through parasitological techniques remains as the gold standard for the diagnosis of trematodiases. In this chapter, we review the current status of knowledge on diagnostic techniques used when examining feces, urine, and sputum and also analyze the most relevant characteristics used to identify eggs with a quick key for the identification of eggs.

Adaptation of the secretome of Echinostoma caproni may contribute to parasite survival in a Th1 milieu.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, broadly employed to study the host-dependent mechanisms that govern the evolution of intestinal helminth infections. Resistance against E. caproni homologous secondary infections has been reported in mice and appears to be related to the generation of a local Th2 response, whereas Th1 responses promote the development of chronic primary infections. Herein, the ability of E. caproni to modulate its secretome according to the host environment is investigated. A two-dimensional differential in gel electrophoresis (2D-DIGE) analysis was performed to elucidate changes in the excretory/secretory products of E. caproni adults after primary and secondary infections in mice. A total of 16 protein spots showed significant differences between groups, and 7 of them were successfully identified by mass spectrometry. Adult worms exposed to a primary infection appear to upregulate proteins involved in detoxification (aldo-keto reductase), stress response (GroEL), and enhancement of parasite survival (acetyl-CoA A-acetyltransferase and UTP-glucose-1-phosphate urydyltransferase). In contrast, any protein was found to be significantly upregulated after secondary infection. Upregulation of such proteins may serve to withstand the hostile Th1 environment generated in primary infections in mice. These results provide new insights into the resistance mechanisms developed by the parasites to ensure their long-term survival.

Antibody trapping: A novel mechanism of parasite immune evasion by the trematode Echinostoma caproni.

BACKGROUND: Helminth infections are among the most prevalent neglected tropical diseases, causing an enormous impact in global health and the socioeconomic growth of developing countries. In this context, the study of helminth biology, with emphasis on host-parasite interactions, appears as a promising approach for developing new tools to prevent and control these infections. METHODS/PRINCIPAL FINDINGS: The role that antibody responses have on helminth infections is still not well understood. To go in depth into this issue, work on the intestinal helminth Echinostoma caproni (Trematoda: Echinostomatidae) has been undertaken. Adult parasites were recovered from infected mice and cultured in vitro. Double indirect immunofluorescence at increasing culture times was done to show that in vivo-bound surface antibodies become trapped within a layer of excretory/secretory products that covers the parasite. Entrapped antibodies are then degraded by parasite-derived proteases, since protease inhibitors prevent for antibody loss in culture. Electron microscopy and immunogold-labelling of secreted proteins provide evidence that this mechanism is consistent with tegument dynamics and ultrastructure, hence it is feasible to occur in vivo. Secretory vesicles discharge their content to the outside and released products are deposited over the parasite surface enabling antibody trapping. CONCLUSION/SIGNIFICANCE: At the site of infection, both parasite secretion and antibody binding occur simultaneously and constantly. The continuous entrapment of bound antibodies with newly secreted products may serve to minimize the deleterious effects of the antibody-mediated attack. This mechanism of immune evasion may aid to understand the limited effect that antibody responses have in helminth infections, and may contribute to the basis for vaccine development against these highly prevalent diseases.

Th2 and Th1 Responses: Clear and Hidden Sides of Immunity Against Intestinal Helminths.

Intestinal helminthiases affect millions of people worldwide, mainly in developing regions, where they cause a significant negative impact on human health and socioeconomic growth of affected populations. However, intestinal helminthiases are still among the most neglected tropical diseases. Protective immunity against intestinal helminths is associated with development of type 2 responses. Nevertheless, in some host-intestinal helminth combinations, local Th1 responses are initiated, inducing chronicity. The usage of helminth-mouse models is useful for elucidating the mechanisms behind the initiation of each type of response. Herein, the current knowledge on these topics is reviewed, paying particular attention to the earliest events of the immune cascade which eventually lead to either susceptibility or resistance to infection.

Interleukin-25 Induces Resistance Against Intestinal Trematodes.

Echinostoma caproni is an intestinal trematode that has been extensively used as an experimental model to investigate the factors determining the resistance to intestinal helminths or the development of chronic infections. ICR mice are permissive hosts for E. caproni in which chronic infections are developed, concomitantly with local Th1 responses, elevated levels of local IFN-γ, inflammation and antibody responses. However, mice develop partial resistance to homologous challenge infections after cure of a primary infection, which converts this subject into an adequate model for the study of the mechanisms generating resistance against intestinal helminths. The purpose of the present study was to compare the immune response induced in primary and secondary infections to elucidate the factors determining the different outcome of the infection in each type of infection. The results obtained indicate that susceptibility is determined by the lack of IL-25 expression in response to primary infection. In contrast, infection in an environment with elevated levels of IL-25, as occurs in challenge infection, results in a Th2 phenotype impairing parasite survival. This was confirmed by treatment of naïve mice with exogenous IL-25 and subsequent infection. Changes induced in goblet cell populations and mucin glycosylation could be implicated in resistance to infection.

Resistance against Echinostoma caproni (Trematoda) secondary infections in mice is not dependent on the ileal protein production.

UNLABELLED: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, which has been widely employed to investigate the factors determining the rejection of intestinal helminths. Protein production patterns of intestinal epithelial cells are related to the infection-induced changes that determine the course of E. caproni infections. Herein, we compare the protein production profiles in the ileum of four experimental groups of mice: control; infected; dewormed and reinfected. Worm burdens were significantly lower in secondary infections, confirming the generation of partial resistance to homologous secondary infections in mice. However, quantitative comparison by 2D-DIGE showed that the protein production profile is similar in control and dewormed mice, and after primary and secondary E. caproni infections. These results showed that, unexpectedly, protein production changes in E. caproni infections are not responsible of resistance development. Fifty-one protein spots were differentially produced between control/treated and infected/reinfected mice and 37 of them were identified by mass spectrometry. The analysis of differentially abundant proteins indicate that cell metabolism and the regulation of proliferation and cell death are the most affected processes after primary and secondary E. caproni infections. These results provide new insights into the proteins involved in the regulation of tissue homeostasis after intestinal infection. SIGNIFICANCE: Intestinal helminthiases are highly prevalent parasitic infections with about 1 billion people infected worldwide. In this scenario, better understanding of host-parasite relationships is needed to elucidate the factors that determine intestinal helminth rejection. The intestinal trematode Echinostoma caproni has been broadly employed in this field, with resistance against secondary homologous infections reported in mice. In this paper, new insights are provided in the regulation of tissue homeostasis after intestinal infection. The unexpected lack of an altered pattern of ileal protein production associated to resistance development suggests that this resistance depends on rapid changes, affecting the early establishment of worms, rather than the activation of later effector mechanisms. These results may contribute to the development of new control tools for the management of these parasitic infections.

Definitive host influences the proteomic profile of excretory/secretory products of the trematode Echinostoma caproni.

BACKGROUND: Echinostoma caproni is an intestinal trematode extensively used as experimental model for the study of factors that determine the course of intestinal helminth infections, since this markedly depends on the host species. Although the host-dependent mechanisms for either chronic establishment or early parasite rejection have been broadly studied, little is known regarding the parasite response against different host environments. METHODS: To identify host-dependent differentially expressed proteins, a comparative proteomic analysis of the excretory/secretory products released from E. caproni adults, isolated from hosts displaying different compatibility with this trematode, was performed. RESULTS: A total of 19 differential protein spots were identified (14 overexpressed in mice and 5 overexpressed in rats). The establishment of chronic infections in mice is mainly associated with the overexpression by adult worms of antioxidant and detoxifying enzymes (e.g. glutathione S-transferase, hydroxyacylglutathione hydrolase, thiopurine S-transferase, etc.) and metabolic enzymes like enolase, leucine aminopeptidase or malate dehydrogenase. However, the overexpression of cathepsin L and the structural protein actin observed in worms isolated from rats seems not to be effective for the colonization of the intestinal mucosa of this host. CONCLUSIONS: The observed differences suggest that protein expression and/or release is modulated by the local environment generated inside the host and provide useful insights in regards to the resistance mechanisms developed by parasites to ensure their long-term survival.

Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda).

BACKGROUND: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. METHODOLOGY/PRINCIPAL FINDINGS: To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. CONCLUSION/SIGNIFICANCE: Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection.