Mathematical algorithm for the automatic recognition of intestinal parasites.

Parasitic infections are generally diagnosed by professionals trained to recognize the morphological characteristics of the eggs in microscopic images of fecal smears. However, this laboratory diagnosis requires medical specialists which are lacking in many of the areas where these infections are most prevalent. In response to this public health issue, we developed a software based on pattern recognition analysis from microscopi digital images of fecal smears, capable of automatically recognizing and diagnosing common human intestinal parasites. To this end, we selected 229, 124, 217, and 229 objects from microscopic images of fecal smears positive for Taenia sp., Trichuris trichiura, Diphyllobothrium latum, and Fasciola hepatica, respectively. Representative photographs were selected by a parasitologist. We then implemented our algorithm in the open source program SCILAB. The algorithm processes the image by first converting to gray-scale, then applies a fourteen step filtering process, and produces a skeletonized and tri-colored image. The features extracted fall into two general categories: geometric characteristics and brightness descriptions. Individual characteristics were quantified and evaluated with a logistic regression to model their ability to correctly identify each parasite separately. Subsequently, all algorithms were evaluated for false positive cross reactivity with the other parasites studied, excepting Taenia sp. which shares very few morphological characteristics with the others. The principal result showed that our algorithm reached sensitivities between 99.10%-100% and specificities between 98.13%- 98.38% to detect each parasite separately. We did not find any cross-positivity in the algorithms for the three parasites evaluated. In conclusion, the results demonstrated the capacity of our computer algorithm to automatically recognize and diagnose Taenia sp., Trichuris trichiura, Diphyllobothrium latum, and Fasciola hepatica with a high sensitivity and specificity.

A simplified method to estimate Diphyllobothrium spp. infection in salmonids.

Some fish parasites constitute severe management problems as they may cause mortality of their fish host or are important zoonoses of humans. Parasite assessments are therefore critical to keep track of infections. If conventional sampling techniques can be simplified, parasite assessments might be easier to obtain, less time-consuming and more extensive. In this study, we compare the assessed number of Diphyllobothrium spp. cysts (CYST) with the counted number of Diphyllobothrium spp. plerocercoid larvae recovered using a conventional digestive technique (LARV). The aim was to determine the potential of using CYST as a simplified methodology for assessing Diphyllobothrium spp. infection in salmonids. In total, 365 brown trout and 424 Arctic charr were sampled from nine lakes in subarctic Norway. Strong correlation, significant linear relationship and large amount of explained variation were found between log10 CYST and log10 LARV in both fish species. The method had a slight, but not significant tendency to work better in charr compared to trout. In addition, absolute difference between CYST and LARV increased at parasite intensities >100 indicating that the method has reduced functionality when estimating parasite intensity in heavily infected salmonid populations. However, overall, using this simplified and less time-consuming methodology, a good indication of Diphyllobothrium spp. intensity, abundance and prevalence was obtained. We suggest that this method provides a sound proxy of the Diphyllobothrium spp. burden and have the potential to be used in parasite assessment during fish monitoring and fisheries management surveys, particularly if the time and resources for detailed parasite studies are not available.

Diphyllobothrium dendriticum (Cestoda: Diphyllobothriidae) in the Intestinal Tract of the Herring Gull Larus argentatus: Localization and Trophic Parameters.

We studied the activities both of digestive enzymes in the small intestine of the herring gull (Larus argentatus) and a tapeworm Diphyllobothrium dendriticum (Cestoda: Diphyllobothriidae) residing in the intestine. It was found that D. dendriticum infects the medial section of the small intestine of the herring gull. Such localization of D. dendriticum is caused by the maximal activity of proteases and glycosidases and by the high rate of membrane and cavitary digestion in this section. The activity of protease and glycosidase in gulls infected with D. dendriticum is decreased. The activity of proteases in the fractions desorbed from the tegument surface of D. dendriticum is significantly higher than that of glycosidases.

Pacific Broad Tapeworm Adenocephalus pacificus as a Causative Agent of Globally Reemerging Diphyllobothriosis.

The Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum) is the causative agent of the third most common fish-borne cestodosis among humans. Although most of the nearly 1,000 cases among humans have been reported in South America (Peru, Chile, and Ecuador), cases recently imported to Europe demonstrate the potential for spread of this tapeworm throughout the world as a result of global trade of fresh or chilled marine fish and travel or migration of humans. We provide a comprehensive survey of human cases of infection with this zoonotic parasite, summarize the history of this re-emerging disease, and identify marine fish species that may serve as a source of human infection when eaten raw or undercooked.

Diphyllobothrium sp. en Canis familiares de la región del subtrópico argentino (Puerto Iguazú, Misiones) / Diphyllobothrium sp. in Canis familiaris from the subtropical area of Argentina (Puerto Iguazú, Misiones)

En este trabajo se informa el hallazgo de huevos de Diphyllobothrium sp. en ejemplares de Canis familiaris (perro doméstico) de Puerto Iguazú, una ciudad subtropical de la provincia de Misiones, Argentina. Durante 2013, en el marco de un relevamiento de la fauna parasitológica de los perros de Puerto Iguazú, se detectaron dos casos positivos en la búsqueda de huevos de Diphyllobothrium sp. La materia fecal de los perros fue recolectada en frascos con formol al 10% y procesada mediante las técnicas de sedimentación de Telemann y de flotación de Sheather. Dado que Misiones no forma parte de la zona endémica de difilobotriasis y considerando, además, su ubicación fronteriza, este hallazgo reviste gran importancia para la salud pública. Se señala la necesidad de actualizar el estado de conocimiento sobre el ciclo de vida de estos parásitos identificando el rango de hospederos intermediarios y definitivos, su potencial zoonótico y la situación epidemiológica en áreas no endémicas

This paper reports the first finding of Diphyllobothrium sp. eggs in Canis familiaris (domestic dog) from Puerto Iguazú, a subtropical city of Misiones province, Argentina. In 2013, two positive cases of Diphyllobothrium sp. eggs were detected during an annual parasitological survey of dogs. Dog feces were collected in vials containing 10% formalin and processed using Telemann's sedimentation and Sheather's flotation techniques. The two cases were detected in rural areas of the municipality. Since Misiones is not a part of the endemic area of diphyllobothriasis and given the fact that it is located in the three-border area of Argentina, Brazil and Paraguay, we consider this finding of great importance to public health. We stress the need for updating the current knowledge about the life cycle of these parasites considering the range of intermediate and definitive hosts, their zoonotic potential, and the epidemiological situation in non-endemic areas

Evaluation of Modified Formalin-Ether Concentration Method Using Para Tube in Clinical Settings.

Conventional formalin-ether concentration method is a gold standard for the diagnosis of parasite infection. However, it may be time-consuming and laborious. We aimed to reveal the clinical usefulness of a modified formalin-ether concentration method using the Para Tube (KS Corporation, Korea) compared with the conventional method. A total of 117 fresh, unpreserved fecal samples composed to 90 negative controls and 27 positive controls with ova of Diphyllobothrium latum/D. nihonkaiense, ova of Clonorchis sinensis and cysts of Giardia lamblia were used in this study. Both methods showed comparable correct identification rate (87.2% for the Para Tube vs. 86.3% for the conventional method).When five samples were examined at once, the Para Tube method reduced the procedure time compared with the conventional method (19 min 58 sec vs. 23 min 18 sec, P=0.0286). We concluded that the modified formalin-ether concentration method using the Para Tube is a rapid, simple, and reliable fecal concentration method for clinical use.

First study of vitellogenesis of the broad fish tapeworm Diphyllobothrium latum (Cestoda, Diphyllobothriidea), a human parasite with extreme fecundity.

In the present study, the process of vitellogenesis of one of the most prolific organisms, the broad tapeworm, Diphyllobothrium latum, the causative agent of human diphyllobothriosis, was studied for the first time using transmission electron microscopy. Cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for detection of glycogen was applied. Starting from the periphery toward the center of the vitelline follicle four stages of vitellocytes are differentiated: immature vitellocytes, early maturing vitellocytes, advanced maturing and mature vitellocytes. Differentiation into mature vitellocytes involves the formation of shell globule clusters containing shell globules, large amount of saturated lipid droplets and glycogen. A peculiar ultrastructural feature of D. latum vitellogenesis is the presence of lamellar bodies in the cytoplasm of mature vitellocytes. This feature is similar to that present in the closely related caryophyllideans and spathebothriideans. Despite the great similarity observed in the embryonic development of diphylobothriideans, caryophyllideans and spathebothriideans, and the fact that their vitellocytes share a feature not reported from other cestode groups, there are substantial differences in the morphology of vitelline clusters, types, amount and localization of their nutritive reserves.

[The specific features of Diphyllobothrium latum circulation in the natural foci of the Kostroma region].

Three hundred pikes in the Kostroma section of the Gorky Reservoir, in the Kostroma Overflow, and in the Galich and Chukloma Lakes were examined for infection with Diphyllobothrium latum larvae. In the Kostroma section of the Gorky Reservoir and in the Kostroma Overflow, diphyllobothriasis was recorded in 66.7% (12/18) and 14.7% (11/75) of the pikes, respectively. The high intensity of the epidemic process in diphyllobothriasis was confirmed in the Kostroma Region. The number of notified cases was noted to tend to increase: 35 and 173 cases in 1994 and 1999, respectively.

[Morphofunctional characteristics of thymus and immunological parameters in golden hamster under experimental invasion with the cestode Diphyllobothrium dendriticum].

Morphofunctional changes induced by the tapeworm in the thymus and blood of its definitive host, by an example of the experimental model "Diphyllobothrium dendriticum--Mesocricetus auratus", have been investigated. The morphofunctional changes in the thymus and immunological transformations in the organism of infested hamster are evidences of rather high immunogenic effect of the D. dendriticum antigens on the definitive host at the period of active growth of the parasites.

Detection and identification of Diphyllobothrium nihonkaiense plerocercoids from wild Pacific salmon (Oncorhynchus spp.) in Japan.

We investigated the risk of diphyllobothriasis from ingestion of wild Pacific salmon in Japan by surveying Diphyllobothrium plerocercoids in 182 salmon samples obtained from Japan. The plerocercoids were not detected in chum salmon (Oncorhynchus keta) (0/26), called Akizake in Japan, caught between September and November. However, the detection rate of plerocercoids in chum salmon, called Tokishirazu in Japan, caught between early April and June, was 51.1% (24/47) with an average of two plerocercoid larvae per fish. The detection rates of cherry salmon (Oncorhynchus masou) and pink salmon (Oncorhynchus gorbuscha) were 12.2% (10/82) and 18.5% (5/27), respectively, and the average number of plerocercoids per fish was 0.45 (37 larvae/82 fishes) and 0.22 larvae (6 larvae/27 fishes), respectively. Plerocercoids isolated from O. keta and O. masou were identified as Diphyllobothrium nihonkaiense on the basis of molecular analysis of the cox1 and nad3 genes. Moreover, four tapeworms (three from O. keta and one from O. masou) were obtained by infecting golden hamsters with plerocercoids. The morphological features of these tapeworms were similar to those of D. nihonkaiense isolated from humans. Therefore, we think that O. keta and not O. masou is the most important source of plerocercoid infections in Japan.

Imported diphyllobothriasis in Switzerland: molecular evidence of Diphyllobothrium dendriticum (Nitsch, 1824).

We report for the first time in Switzerland a clinical case because of Diphyllobothrium dendriticum, identified by molecular methods. We discuss the potential for this imported species to infect local intermediate hosts and thus to achieve autochthonous cyclic transmission.

A rare case of diphyllobothriasis from Pondicherry, South India.

Diphyllobothriasis is an intestinal parasitic infection caused by the ingestion of raw fresh-water fish containing the infectious larvae of Diphyllobothrium spp. This infection is uncommon in India. We report a case of diphyllobothriasis that occurred in Pondicherry, India, in a 5-year-old boy hailing from a fishing community. He attended the Pediatric OPD with spontaneous discharge of segments of the adult parasite. The segments (macroscopically and microscopically) were identified as those of Diphyllobothrium latum. The stool examination also revealed characteristic oval eggs.

Diphyllobothrium nihonkaiense (Yamane et al., 1986) in Switzerland: first molecular evidence and case reports.

We report the first cases of locally-acquired Diphyllobothrium nihonkaiense (Yamane, Kamo, Bylund and Wikgren, 1986) in Switzerland, confirmed by genetic analysis (18S rRNA, COI and ITS1-5.8S rRNA-ITS2 genes). Diphyllobothriasis in this country is attributed to the tapeworm D. latum (Linnaeus, 1758) but the increasing popularity of raw fish culinary specialities (sushi, carpaccio, tartare) brings out a new diagnostic problem, so that people can get infected by exotic species of tapeworms.

Diphyllobothriasis, Brazil.

Cases of human diphyllobothriasis have been reported worldwide. Only 1 case in Brazil was diagnosed by our institution from January 1998 to December 2003. By comparison, 18 cases were diagnosed from March 2004 to January 2005. All patients who became infected ate raw fish in sushi or sashimi.

[Infestation with the tapeworm Diphyllobothrium latum after eating raw fish]. / Infestatie met de lintworm Diphyllobothrium latum na het eten van rauwe vis.

A 31-year-old man with no relevant medical history encountered a white, ribbon-shaped object, 15 cm long and approximately 1 cm wide, in his faeces. It turned out to be Diphyllobothrium latum, a tapeworm that has fish as the intermediate host. The patient had eaten raw fish and shellfish during a holiday in Brazil 5 months before. He recovered after a single dose of praziquantel.

[The history of the exploration of the Diphyllobothrium latum life cycle]. / Historia odkrycia i opisania cyklu rozwojowego Diphyllobothrium latum L.

Diphyllobothrium latum typically occur in the subarctic and temperature zones of the Eurasian Continent, however it is also observed in the Arctic Region and Australia. Raw fish meat and raw shell-fish containing plerocercoids is the main source of human infections. Humans are the principial final host of D. latum. Under primitive conditions human excrements with immature tapeworm eggs are deposited anywhere. After embrionic development which occurs in the water, the eggs are transformed into coracidia. Two intermediate hosts (cyclop and fish) are needed for further development. The free-swimming coracidium must enter the stomach of different species of Copepoda. After contact with the intestinal juice of the cyclop, the coracidium loses its ciliated envelope and the hooks become mobile. The metamorphosis of the oncosphere to the procercoid occurs. The discovery of the procercoid done by Janicki and Rosen in 1917 implied that the missing link in the cycle of the broad tapeworm had been found. The copepod including the procercoid is consumed by the second intermediate host. The larvae escape from the digestive tract of the fish and are transformed into a plerocercoid. The plerocercoids develop slowly in the fish, and they must reach a certain degree of maturity to be capable of infecting the final host. Today the life cycle of the tapeworm is well recognized and well illustrated in parasitological textbooks and websites. In this paper the history of the exploration of the D. latum life cycle is described. Additionally the main scientific researches carried out on life stages of the broad tapeworm have been reviewed.

Current situation of human diphyllobothriasis in Europe.

Diphyllobothriasis, a parasitosis caused by the flatworm Diphyllobothrium latum, is contracted by consuming raw or undercooked freshwater fish. The aim of this study was to evaluate the situation of this parasitosis during the past 20 years in Europe through the analysis of databases and search engines (Medline, Cabi Helminthological abstracts,Yahoo, Google), and through a questionnaire sent to a network of European parasitologists and to microbiological laboratories located on the shores of the large Alpine lakes. This study has shown that several dozen cases have been reported each year in Finland and Sweden, that there have been numerous cases in the French or Italian speaking areas of subalpine lakes, and that sporadic cases only have been observed in Austria, Spain, Greece, Romania, Poland and Norway. Over 30 cases have been identified on the Swiss shores of Lake Maggiore since 1990, and 70 cases on the Swiss and French shores of Lake Leman between 1993 and 2002. Eight to 12% of perch fillets from Lake Leman and 7.8 % of perch from Lake Maggiore were infested with larvae. Contamination sources include marinated fish fillets in northern Europe, 'carpaccio di persico' in northern Italy, and perch and charr consumed raw or undercooked around Lake Leman. Factors allowing the continuation of the parasitic cycle include the continued dumping of wastewater into lakes, yachtsmen who also fish, and a possible animal reservoir.

Chemical structures and immunolocalization of glycosphingolipids isolated from Diphyllobothrium hottai adult worms and plerocercoids.

Glycosphingolipids (GSLs) were purified from adults and plerocercoids of the tapeworm Diphyllobothrium hottai, and their chemical structures were determined. Total lipid fractions prepared from chloroform/methanol extracts of whole tissues were fractionated successively on ion-exchange chromatography, silicic acid column chromatography, and preparative TLC. The purified GSLs were characterized by methylation analysis, TLC-immunostaining, liquid secondary ion MS, MALDI-TOF MS, and 1H-NMR. Ten GSLs were isolated from adult worms and four from plerocercoids, comprising mono-, di-, tri-, tetra-, and pentasaccharides. The GSL Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta 1-Cer was found in adult worms but not in plerocercoids, whereas Ga lbeta 1-4 (Fuc alpha 1-3)Glc beta 1-3(Gal beta 1-6)Gal beta 1-Cer was found in both adult worms and plerocercoids. We previously found a similar series of GSLs in plerocercoids of the cestode Spirometra erinaceieuropaei, and termed them 'spirometosides'[Kawakami, Y. et al. (1996) Eur J. Biochem. 239, 905-911]. The core structure of spirometosides, Gal beta 1-4Glc beta 1-3 Gal beta 1-Cer, may have taxonomic significance, being characteristic of pseudophyllidean tapeworms. In the present study, GSL compositions were significantly different between adults and plerocercoids, and growth-dependent changes in composition were documented. We found a novel dihexosylceramide, Glc beta 1-3Gal beta 1-Cer, which is a possible precursor for spirometosides. Immunohistochemical examination showed that spirometoside GSLs are highly enriched in the inner surface of bothria, the major point of contact between the adult worm and the host's intestine. Our findings indicate that spirometosides are involved in host-parasite interaction.

Five cases of Diphyllobothrium nihonkaiense infection with discovery of plerocercoids from an infective source, Oncorhynchus masou ishikawae.

Five persons from 2 families residing at Miyama Town, Mie Prefecture, Japan, ingested fresh raw fish Oncorhynchus sp. on 9 May 1999 that was caught at Owase district in Mie. They all expelled diphyllobothriid cestodes 11-37 days after ingesting the fish. The parasites were morphologically identical to Diphyllobothrium nihonkaiense Yamane et al., 1986. Five plerocercoids were detected from a portion of the fish. Nucleotide sequence of a region of the cytochrome c oxidase subunit I gene of mitochondrial DNA from an adult worm was identical with that from the plerocercoid. The fish was identified as Oncorhynchus masou ishikawae according to the nucleotide sequence of the nuclear ribosomal second internal transcribed spacer region II gene. This is the first record of D. nihonkaiense plerocercoids from O. m. ishikawae.