Real-time qPCR coupled with high-resolution melting curve analysis for the detection of the internal transcribed spacer 1 of Angiostrongylus costaricensis.

Abdominal angiostrongyliasis (AA) is a zoonotic and severe parasitic infection caused by Angiostrongylus costaricensis. AA is currently diagnosed by the observation of A. costaricensis-compatible structures in biopsies or the detection of antibodies in serological tests. However, molecular methods targeting homologous sequences of A. costaricensis have not been designed before, and therefore, an HRM-coupled qPCR was developed to detect the internal transcribed spacer 1 (ITS1) of the parasite. The present assay successfully amplified DNA of A. costaricensis obtained from different hosts and identified slight sequence differences through the HRM analysis. The detection limit of the HRM-qPCR was 0.00036 ng/µL, 1.0 ng/µL, and 0.1 ng/µL when A. costaricensis DNA was diluted in nuclease-free water, whole blood, and sera, respectively, which highlights its potential use for cell-free DNA detection. Moreover, the reaction did not cross-amplify DNA of Angiostrongylus cantonensis, Strongyloides stercoralis, and other nematodes, thus emphasizing its specificity. Additionally, the assay tested positive in formalin-fixed paraffin embedded biopsies with visible A. costaricensis adults or eggs, but not in samples without evident parasites or a low number of larvae, which suggests that the reaction is useful for confirming the presence of the nematode in clinical samples. Finally, DNA of sera from patients with AA was evaluated with the HRM-qPCR but none tested positive, possibly due to long storage periods of the samples which could have led to cfDNA degradation. These results indicate that this assay may be useful in the confirmation of AA and its prospection for cell-free DNA detection protocols.

Paronatrema davidbowiei n. sp. (Trematoda: Syncoeliidae), a new parasite of the pelagic thresher (Alopias pelagicus) and its phylogenetic relationships within the suborder Hemiurata Skrjabin & Guschanskaja, 1954.

A new species of hemiurid trematode found on the gills and in the aorta of the pelagic thresher Alopias pelagicus from the eastern Pacific, off Costa Rica, is described based on an integrative taxonomic approach that includes the use of light and scanning electron microscopy, and 28S rDNA sequencing. Phylogenetic analysis was also performed to explore, for the first time, the relationships of a member of the subfamily Otiotrematinae within the suborder Hemiurata. Paronatrema davidbowiei n. sp. can be distinguished from the congeners by having tegumental spines on the dorsal surface of the forebody, papillae on the oral sucker, and different morphology or number of testicular follicles. BLAST analysis revealed that sequences of Paronatrema davidbowiei n. sp. had the highest degree of similarity with Hirudinella spp. (Hirudinellidae). Results from Maximum Likelihood and Bayesian phylogenetic analyses, returning trees with the exact same topology and strong branch support, distinguished between the two superfamilies included in the suborder Hemiurata: Azygioidea and Hemiuroidea. Our analysis placed the new species in a clade with Copiatestes filiferus, the only existing sequence of the family Syncoeliidae.

Seek, and you will find: Cryptic diversity of the cardiopulmonary nematode Angiostrongylus vasorum in the Americas.

Angiostrongylus vasorum is a metastrongylid parasite infecting wild canids and domestic dogs. Its patchy distribution, high pathogenicity and taxonomical classification makes the evolutionary history of A. vasorum intriguing and important to study. First larval stages of A. vasorum were recovered from feces of two grey foxes, Urocyon cinereoargenteus, from Costa Rica. Sequencing and phylogenetic and haplotypic analyses of the ITS2, 18S and cytochrome oxidase subunit 1 (cox1) fragments were performed. Then p- and Nei´s genetic distance, nucleotide substitution rates and species delimitation analyses were conducted with cox1 data of the specimens collected herein and other Angiostrongylus spp. Cophylogenetic congruence and coevolutionary events of Angiostrongylus spp. and their hosts were evaluated using patristic and phenetic distances and maximum parsimony reconciliations. Specimens from Costa Rica clustered in a separate branch from European and Brazilian A. vasorum sequences in the phylogenetic and haplotype network analyses using the ITS2 and cox1 data. In addition, cox1 p-distance of the sequences derived from Costa Rica were up to 8.6 % different to the ones from Europe and Brazil, a finding mirrored in Nei´s genetic distance PCoA. Species delimitation analysis supported a separate group with the sequences from Costa Rica, suggesting that these worms may represent cryptic variants of A. vasorum, a new undescribed taxon or Angiocaulus raillieti, a synonym species of A. vasorum described in Brazil. Moreover, nucleotide substitution rates in A. vasorum were up to six times higher than in the congener Angiostrongylus cantonensis. This finding and the long time elapsed since the last common ancestor between both species may explain the larger diversity in A. vasorum. Finally, cophylogenetic congruence was observed between Angiostrongylus spp. and their hosts, with cospeciation events occurring at deeper taxonomic branching of host order. Altogether, our data suggest that the diversity of the genus Angiostrongylus is larger than expected, since additional species may be circulating in wild canids from the Americas.

Zoonotic Mansonella ozzardi in Raccoons, Costa Rica, 2019-2022.

Mansonella ozzardi, a filarioid parasite, causes human mansonellosis in the Americas. We identified raccoons (Procyon lotor) as wildlife reservoirs of M. ozzardi in Costa Rica. Noting the sympatry of free-ranging raccoons and humans, we conclude that mansonellosis is a considerable public health risk in the region.

Molecular, morphological and histopathological evidence of Spirometra mansoni in wild and domestic animals from Costa Rica.

Spirometra mansoni is a diphyllobothroid cestode and one of the causing agents of sparganosis, a zoonotic foodborne and waterborne infection in humans. This parasite has an indirect life cycle with domestic and wild canids or felids as definitive hosts. The last report of S. mansoni in Costa Rica was done in 2004 by morphological assessment of worms, whereas molecular evidence of this species was obtained recently in the Americas. Herein, we present seven cases of spirometrosis in four dogs, three cats and a coyote from different regions of Costa Rica occurring in a time span of a year. Dog cases presented vomiting, hyporexia, lethargy and diarrhea, whereas cats were mostly asymptomatic. Moreover, the coyote was found with Spirometra sp. proglottids incidentally. Cytochrome oxidase subunit 1 (cox1) sequences of eggs or proglottids derived from all cases were analyzed with a Bayesian Inference phylogenetic tree and a haplotype network. These analyses showed the clustering of S. mansoni from Costa Rica with other sequences derived from Asia and America. Moreover, cox1 sequences clustered in two separate haplotypes, suggesting the high genetic diversity of the species. The present cases represent the first molecular evidence of the parasite in Central America; thus, extending its known range in the American continent.

Zoonotic Ancylostoma ceylanicum Infection in Coyotes from Guanacaste Conservation Area, Costa Rica, 2021.

Ancylostoma ceylanicum is the second most common hookworm infecting humans in the Asia-Pacific region. Recent reports suggest presence of the parasite in the Americas. We report A. ceylanicum infections in coyotes from the Guanacaste Conservation Area, Costa Rica. Our findings call for active surveillance in humans and animals.

Diagnostic criteria and case definitions for abdominal angiostrongyliasis: a systematic review from the Brazilian experience.

Although rare, Angiostrongylus costaricensis infection may be a more prevalent etiology of inflammatory bowel disease than ulcerative colitis and Chron's disease in endemic areas in Central and South America. The present study reviewed the occurrence of A. costaricensis in Brazil, its clinical presentation and pathology; and proposed diagnostic criteria and case definitions for abdominal angiostrongyliasis (AA). Southern and southeastern Brazilian regions are the main endemic areas, and AA affects both genders and all age groups. A review of all 23 published reports of 51 Brazilian patients highlighted the following features that were subsequently classified as minor diagnostic criteria: abdominal pain, palpable mass in the right lower abdominal quadrant, history of exposure, ileocecal tumor, and intestinal perforation with wall thickening. Proposed major criteria include right lower quadrant abdominal pain, blood eosinophilia, positive serology (antibody detection), intense eosinophilic infiltration that involves all strata of the intestinal wall, eosinophilic granulomatous reaction, and eosinophilic vasculitis. In addition to the definitions of suspected and possible cases according to increasing strength of evidence of this infection, demonstration of worms/eggs/larvae in tissues or Angiostrongylus DNA in tissues or serum are required for a confirmed diagnosis. The application of the proposed criteria and definitions may improve patient management, epidemiologic surveillance, and identification of new endemic areas.

Wildlife parasitology: sample collection and processing, diagnostic constraints, and methodological challenges in terrestrial carnivores.

Wild terrestrial carnivores play a crucial role as reservoir, maintenance, and spillover hosts for a wide parasite variety. They may harbor, shed, and transmit zoonotic parasites and parasites of veterinary importance for domestic hosts. Although wild carnivores are globally distributed and comprise many different species, some living in close proximity to human settlements, only a few studies have investigated parasites of wild terrestrial carnivores using non-specific techniques. Access to samples of wild carnivores may be challenging as some species are protected, and others are secretive, possibly explaining the data paucity. Considering the importance of wild carnivores' health and ecological role, combined with the lack of specific diagnostic methodologies, this review aims to offer an overview of the diagnostic methods for parasite investigation in wild terrestrial carnivores, providing the precise techniques for collection and analysis of fecal, blood, and tissue samples, the environmental impact on said samples, and the limitations researchers currently face in analyzing samples of wild terrestrial carnivores. In addition, this paper offers some crucial information on how different environmental factors affect parasite detection postmortem and how insects can be used to estimate the time of death with a specific highlight on insect larvae. The paper contains a literature review of available procedures and emphasizes the need for diagnostic method standardization in wild terrestrial carnivores.

Integrative taxonomy of Anaporrhutum mundae sp. nov. (Trematoda: Gorgoderidae), a parasite of the Munda round ray Urotrygon munda (Urotrygonidae) in Costa Rica.

A new species of trematode of anaporrhutine gorgoderid, from the gill chambers of the Munda round ray Urotrygon munda in Costa Rica is described, based on an integrative taxonomic approach that includes the use of light and scanning electron microscopy, ITS2 and 28S rDNA sequencing, and phylogenetic analysis. Anaporrhutum mundae sp. nov. can be distinguished from congeneric species by a combination of morphological traits and particularly by having the genital pore opening at the level of the intestinal bifurcation. The new species also can be distinguished from all other species of Anaporrhutum, except A. euzeti Curran, Blend & Overstreet, 2003, by having fewer testicular follicles per testis. Anaporrhutum mundae sp. nov. also differs from A. euzeti in its forebody shape and by having different morphology and location of the vitellaria. The study of the tegumental surface of A. mundae sp. nov., as revealed by scanning electron microscopy, allowed detection of new morphological characters for a member of Anaporrhutinae that may be of taxonomic value. These are: a stylet cavity dorsal to the oral sucker with a large penetration gland opening on each side of the cavity and small penetration gland openings located ventral to the stylet cavity, arranged in a circle around the mouth. This represents the first record of an Anaporrhutum species from Costa Rica. Further, A. mundae sp. nov. represents the first parasite described or reported in this host.

Case report: First documented case of cerebral angiostrongyliasis caused by Angiostrongylus costaricensis in a free-ranging opossum.

Angiostrongylus costaricensis is a metastrongyloid nematode that primarily infects the mesenteric arteries of wild rodents. This parasite is endemic in several regions of the American continent, and in humans, causes a disease known as abdominal angiostrongyliasis. Despite the important health implications of this nematode, there are limited studies investigating the involvement of wild animals in its life cycle. In this study, we present the clinical manifestations, pathologic findings, and molecular diagnosis, to the best of our current knowledge, of the first documented onset of cerebral angiostrongyliasis because of A. costaricensis infection in a juvenile free-ranging opossum (Didelphis marsupialis). Histopathological findings stress the presence of eosinophilic meningoencephalitis with nematodes present within the lesions, and PCR was positive for cox1 and ITS1 reactions. The obtained sequences for a 279 bp fragment of ITS1 were 100% identical to A. costaricensis from Costa Rica. This case highlights the substantial difficulties in diagnosing neuroangiostrongyliasis, yet underscores the importance of considering A. costaricensis as a potential culprit behind neurological conditions in wild marsupials. It acts as an urgent call to action to improve surveillance programs tracking infectious and parasitic diseases causing mortality in wildlife populations.

Mitochondrial and ribosomal markers in the identification of nematodes of clinical and veterinary importance.

BACKGROUND: Nematodes of the Ascarididae, Ancylostomatidae and Onchocercidae families are parasites of human and veterinary importance causing infections with high prevalence worldwide. Molecular tools have significantly improved the diagnosis of these helminthiases, but the selection of genetic markers for PCR or metabarcoding purposes is often challenging because of the resolution these may show. METHODS: Nuclear 18S rRNA, internal transcribed spacers 1 (ITS-1) and 2 (ITS-2), mitochondrial gene cytochrome oxidase 1 (cox1) and mitochondrial rRNA genes 12S and 16S loci were studied for 30 species of the mentioned families. Accordingly, their phylogenetic interspecies resolution, pairwise nucleotide p-distances and sequence availability in GenBank were analyzed. RESULTS: The 18S rRNA showed the least interspecies resolution since separate species of the Ascaris, Mansonella, Toxocara or Ancylostoma genus were intermixed in phylogenetic trees as opposed to the ITS-1, ITS-2, cox1, 12S and 16S loci. Moreover, pairwise nucleotide p-distances were significantly different in the 18S compared to the other loci, with an average of 99.1 ± 0.1%, 99.8 ± 0.1% and 98.8 ± 0.9% for the Ascarididae, Ancylostomatidae and Onchocercidae families, respectively. However, ITS-1 and ITS-2 average pairwise nucleotide p-distances in the three families ranged from 72.7% to 87.3%, and the cox1, 12S and 16S ranged from 86.4% to 90.4%. Additionally, 2491 cox1 sequences were retrieved from the 30 analyzed species in GenBank, whereas 212, 1082, 994, 428 and 143 sequences could be obtained from the 18S, ITS-1, ITS-2, 12S and 16S markers, respectively. CONCLUSIONS: The use of the cox1 gene is recommended because of the high interspecies resolution and the large number of sequences available in databases. Importantly, confirmation of the identity of an unknown specimen should always be complemented with the careful morphological examination of worms and the analysis of other markers used for specific parasitic groups.

ParSCo: celebrating 10 years of a unique parasitology summer course.

ParSCo (Parasitology Summer Course) is an intense, 1-week-long summer course organized by the Parasitology Unit of the Department of Veterinary Medicine, University of Bari, Italy, with the support of the World Association for the Advancement of Veterinary Parasitology (WAAVP), the European Veterinary Parasitology College (EVPC) and Parasites and Vectors. The course, which is conducted in southern Italy, is planned for parasitologists and post-graduate students working in the field of parasitology. The course consists of theoretical and practical lessons, which include the collection, identification and diagnosis of parasites of pets, livestock and wildlife. The participants in ParSCo are afforded the opportunity to be involved in clinical examination and sample collection for the diagnosis of parasitic diseases (e.g. leishmaniosis, thelaziosis and many tick-borne diseases) present in the Mediterranean Basin. The course is conducted at Casa di Caccia, a hunting lodge situated in the Gallipoli Cognato Forest near the Basento River in the Basilicata region in southern Italy. In addition to the training purpose, ParSCo is a great opportunity for sharing knowledge and expertise while becoming part of the parasitology community in a pleasant environment. In this editorial, we share some information and celebrate 10 years of ParSCo, looking forward to forthcoming sessions of this unique parasitology summer course.

Social media as a tool for detecting underdiagnosed parasitic infections: the case of spirocercosis.

Spirocercosis is a parasitic disease caused by the nematode Spirocerca lupi that affects several canids around the world. In Costa Rica, few spirocercosis cases have been reported; however, it is suspected that the infection is currently underdiagnosed. As seen during the COVID-19 pandemic, social media has sped up the spread of scientific knowledge, raising practioner's awareness of the epidemiology and signs and manifestations of infections. The aim of this work was to use social media as a tool for increasing awareness of spirocercosis among veterinarians and investigate the frequency of cases occurring in Costa Rica. Infographic material about the infection was disseminated by Whatsapp, Facebook, and Instagram from January 2021 to July 2022 using a public account of the Laboratory of Helminthology of the University of Costa Rica. In addition, Google Forms questionnaires were completed by veterinarians to report the number of spirocercosis cases in their clinics, associated manifestations, diagnosis, treatment, and outcome of the disease. Remarkably, since 2020, a six-fold increase in the number of cases per year was noted. Clinical manifestations matched those reported elsewhere, and one of every three dogs infected with S. lupi died spontaneously or required euthanasia because of the infection. This work suggests that S. lupi infection in Costa Rica may have been underdiagnosed in the last decade, and the knowledge and awareness of clinical personnel play a major role in the detection of this and other infectious diseases.

Neuroangiostrongyliasis: Updated Provisional Guidelines for Diagnosis and Case Definitions.

Angiostrongylus cantonensis is the main causative agent for eosinophilic meningoencephalitis in humans. Larvae are rarely found in the cerebral spinal fluid (CSF). Consequently, serology and DNA detection represent important diagnostic tools. However, interpretation of the results obtained from these tools requires that more extensive accuracy studies be conducted. The aim of the present study is to update guidelines for diagnosis and case definitions of neuroangiostrongyliasis (NA) as provided by a working group of a recently established International Network on Angiostrongyliasis. A literature review, a discussion regarding criteria and diagnostic categories, recommendations issued by health authorities in China and an expert panel in Hawaii (USA), and the experience of Thailand were considered. Classification of NA cases and corresponding criteria are proposed as follows: minor (exposure history, positive serology, and blood eosinophilia); major (headache or other neurological signs or symptoms, CSF eosinophilia); and confirmatory (parasite detection in tissues, ocular chambers, or CSF, or DNA detection by PCR and sequencing). In addition, diagnostic categories or suspected, probable, and confirmatory are proposed. Updated guidelines should improve clinical study design, epidemiological surveillance, and the proper characterization of biological samples. Moreover, the latter will further facilitate accuracy studies of diagnostic tools for NA to provide better detection and treatment.

A practical guide for the diagnosis of abdominal angiostrongyliasis caused by the nematode Angiostrongylus costaricensis.

Abdominal angiostrongyliasis (AA) is a severe parasitic infection caused by the nematode Angiostrongylus costaricensis. This disease is characterized by abdominal pain, a strong inflammatory eosinophilic response in the blood and tissues, and eventually intestinal perforation. Diagnosis of AA is challenging since there are no commercially available serological kits for A. costaricensis, and thus, histopathological analysis remains the gold standard. Herein we provide a decision flowchart for clinicians to improve the diagnosis of AA based on a patient's clinical manifestations, laboratory findings, macroscopic observations of the gut lesions, as well as characteristic microscopic alterations in biopsies. A brief discussion of the available polymerase chain reaction and in-house serological methods is also presented. The aim of this mini-review is to improve the diagnosis of AA, which should lead to prompt detection of cases and better estimates of the epidemiology and geographical distribution of A. costaricensis.

Cryptic diversity in a gastrointestinal acanthocephalan of New World primates from Costa Rica.

Prosthenorchis elegans is a worm of the family Archiacanthocephala that infects non-human primates in the Americas, producing an intestinal pathology that may compromise the life of its hosts. Squirrel monkeys, Saimiri oerstedii citrinellus, were found with P. elegans in Costa Rica. Histopathological analysis revealed a severe pyogranulomatous response composed by macrophages, neutrophils, eosinophils, fibroblasts and lymphocytes. Morphological worm analyses revealed 36 hooks in the proboscis distributed in six rows; and total body, hook and lemnisci length were compatible to the original descriptions of P. elegans. In addition, phylogenetic, haplotype network and genetic distance analyses were done on cytochrome oxidase subunit 1, cox1, sequences obtained from the collected specimens. Sequences obtained herein clustered separately with high posterior probabilities in a Bayesian Inference tree and showed 8.12% nucleotide differences when compared to P. elegans from Colombia. This high divergence was confirmed in the TCS network that separated Colombian and Costa Rican sequences by 32 mutational steps, a genetic distance PCA which separated sequences from both geographical locations by 89.5% and an FST value of 0.655, indicating the presence of cryptic diversity in P. elegans. Additional studies from specimens collected from other definitive hosts and geographical locations are required to better understand the biodiversity of this species.

First morphological and molecular report of Lagochilascaris minor (Nematoda, Ascarididae) in a domestic cat from Veracruz, Mexico.

The clinical presentation and the morphological and molecular findings of Lagochilascaris minor in a domestic cat from Veracruz, Mexico are described. In the physical examination of the cat, a fistula with purulent exudate was observed in the ventral middle and left lateral region of the neck, with the presence of nematodes inside. Thirty-two worms were recovered from the lesion, which were identified based on their morphological characteristics as Lagochilascaris spp. To determine the species of the nematode, DNA was extracted from the worms and a fragment of the cytochrome C oxidase subunit 1 was amplified. The product obtained was 100% similar with L. minor from Costa Rica. This is the first morphological and molecular report of L. minor parasitizing a domestic cat from Veracruz, Mexico, and emphasizes the circulation of this zoonotic parasite in the country.

Emerging Lagochilascaris minor infections in domestic cats from Costa Rica: A zoonotic threat for the region.

Two cases of lagochilascariasis minor in domestic cats from Costa Rica within a period of two months are recorded for the first time in Central America. Clinical findings included purulent discharge and a tumor in the left ear in one of the cats, whereas the other cat had an ulcerated cervical lesion. Both patients underwent surgical procedures during which nematode worms were collected and analyzed. The collected nematodes were identified using a combination of morphological and molecular assays, which revealed a 99.1% similarity in the cytochrome oxidase subunit 1 with L. minor from Mexico. The lack of information on this parasitosis, as well as the enormous harm it does to animal and human hosts, highlights the need for more research and awareness in Costa Rica and Central America. Furthermore, the unexpected occurrence of these instances in the same location emphasizes the imminent zoonotic risk to humans and the active circulation of the parasite.

What lies behind the curtain: Cryptic diversity in helminth parasites of human and veterinary importance.

Parasite cryptic species are morphologically indistinguishable but genetically distinct organisms, leading to taxa with unclear species boundaries. Speciation mechanisms such as cospeciation, host colonization, taxon pulse, and oscillation may lead to the emergence of cryptic species, influencing host-parasite interactions, parasite ecology, distribution, and biodiversity. The study of cryptic species diversity in helminth parasites of human and veterinary importance has gained relevance, since their distribution may affect clinical and epidemiological features such as pathogenicity, virulence, drug resistance and susceptibility, mortality, and morbidity, ultimately affecting patient management, course, and outcome of treatment. At the same time, the need for recognition of cryptic species diversity has implied a transition from morphological to molecular diagnostic methods, which are becoming more available and accessible in parasitology. Here, we discuss the general approaches for cryptic species delineation and summarize some examples found in nematodes, trematodes and cestodes of medical and veterinary importance, along with the clinical implications of their taxonomic status. Lastly, we highlight the need for the correct interpretation of molecular information, and the correct use of definitions when reporting or describing new cryptic species in parasitology, since molecular and morphological data should be integrated whenever possible.