Fish-borne trematode metacercariae detected in fish commonly used for raw consumption in Ninh Binh Province, Vietnam.

Raw or undercooked fish dishes are the major sources of human infection of fishborne trematodes (FBT) and the situation of metacercarial infection in fish greatly affect the prevalence in humans, especially those fish that are commonly used for raw consumption. To investigate the situation of infection with metacercaria of FBT in fish often used to prepare raw fish dishes by local people to assess the risk of infection to humans in Ninh Binh province, Vietnam. 345 fish belonging to five species of freshwater and one species of brackish water fish were collected from fishermen or small-scale fish dealers in Kim Son and Yen Khanh districts, Ninh Binh province between May 2017 and May 2018. Metacercaria of FBT was discovered by pepsin and hydrochloric acid digestion techniques and identified by the morphological and molecular analysis. Among examined fish, 44.06% infected with FBT metacercaria and the highest prevalence was in Cyprinus carpio (86.54%), Ctenopharyngodon idellus (78.43%) and Hypophthalmichthys molitrix (66.67%) while Konosirus punctatus - the brackish water fish - were free from infection. Three species of FBT were found; namely Haplorchis pumilio (accounting for 99.84% of collected metacercariae), Haplorchis taichui and Clonorchis sinensis. The average density was 1.06 metacercariae per gram of freshwater fish and the highest number was of C. idellus (6.38 cysts/gram) followed by Cirrhinus molitorella and C. carpio. Results of the study show the high prevalence of infection of FBT metacercariae among freshwater fish often used to prepare raw fish dishes in Ninh Binh province. These findings suggest the need for greater awareness of the risk from raw fish dishes among public health authorities and people.

Evidence of Centrocestus formosanus (Nishigori, 1924) in Zebrafish (Danio rerio).

This study reports the presence of cartilaginous cysts in zebrafish gills, purchased at the end of 2012 before the application of the Italian Legislative Decree 26/2014 and used for experimental research. It highlights the importance of using specific pathogen-free organisms from reliable production establishments to avoid obtaining false results in experiments. Of 20 samples analyzed, 4 showed cysts in gills, with abnormal tissue modification and cartilaginous metaplasia induced by prolonged parasitic insult. The results obtained from the periodic health monitoring program showed the presence of metacercariae of digenean trematodes, specifically identified as Centrocestus formosanus.

New locality record for Haplorchoides mehrai and possible interactions with Opisthorchis viverrini metacercariae in cyprinid fishes in Northeast Thailand.

Metacercariae of Opisthorchis viverrini, a carcinogenic liver fluke, and Haplorchoides sp., a trematode maturing in catfish, are commonly found in cyprinid fish, the second intermediate hosts of both flukes. However, the specific identity of Haplorchoides sp. in Thailand and a precise assessment of the effects of co-infections with O. viverrini have never been clarified. Therefore, we aimed to identify the species of Haplorchoides and to investigate possible interactions of the two trematode species in cyprinid fishes. Based on the morphology and morphometry of the cercaria, metacercaria, and adult stages, the Haplorchoides species found was identified as Haplorchoides mehrai Pande and Shukla 1976. Thailand is formally recorded as a new locality for H. mehrai, where naturally infected hosts include the snail Melanoides tuberculata (first intermediate host), the cyprinid fishes Hampala dispar, Cyclocheilichthys apogon, Puntius leiacanthus, Labiobarbus burmanicus, and Cirrhina jullieni (second intermediate hosts), and a catfish, Mystus nemurus (definitive host). The co-infection rates of O. viverrini and H. mehrai were significantly associated with fish species and fish body region (P < 0.001), with an overall significantly higher average intensity of H. mehrai (126.26 metacercariae/fish) than that of O. viverrini (18.02 metacercariae/fish). Further work is required to demonstrate the extent and mechanisms of possible interactions between these trematode species in the fish host. These data may provide a better understanding of O. viverrini transmission dynamics, and help design integrated control interventions.

Molecular characterization of Stictodora tridactyla (Trematoda: Heterophyidae) from Kuwait Bay using rDNA ITS and mtCO1.

Stictodora tridactyla is an intestinal fluke in the family Heterophyidae that parasitizes shorebirds and mammals, including humans. Its metacercarial cyst stage was reported in the Arabian killifish, Aphanius dispar, at Kuwait Bay. In the present study, Cerithidea cingulata was found to serve as the first intermediate host of S. tridactyla. In order to establish the snail-fish link in the life cycle of S. tridactyla, complete sequences of ribosomal DNA internal transcribed spacer region 1 and 2 (rDNA ITS1 and ITS2) and partial sequence of cytochrome oxidase subunit 1 were obtained for metacercarial cysts isolated from the fish A. dispar and rediae isolated from the snail C. cingulata. Sequence alignment demonstrated that these larval stages belong to the same heterophyid species, S. tridactyla. Phylogenetic analysis based on rDNA ITS1, ITS2, and mtCO1 confirmed the position of S. tridactyla within the Heterophyidae and found it to cluster with Haplorchis spp. The present study represents the first molecular study correlating the larval stages of S. tridactyla using rDNA ITS1, ITS2, and mtCO1 and examining the phylogenetic relationships of S. tridactyla with different heterophyid species.

Heterophyid trematodes recovered from people residing along the Boseong River, South Korea.

We conducted an epidemiological survey to determine the status of heterophyid fluke infections among people residing along the Boseong River, Gokseong-gun, South Korea (= Korea) from October 2011 to February 2012. Fecal specimens were collected from 115 (male 51, female 64) people and examined for intestinal helminth eggs using the Kato-Katz thick smear technique. The eggs of Metagonimus yokogawai together with other Metagonimus spp. were detected in 28 (24.3%) cases. Eleven egg positive people were treated with 10mg/kg praziquantel followed by MgSO4 purging in order to recover the adult flukes. Whole consecutive diarrheic stools were collected individually 4-5 times. Adult flukes recovered were 66,499 specimens (6045.4/positive case) of M. yokogawai, 343 (38.1) of Metagonimus miyatai, 3293 (299.4) of Metagonimus takahashii, 81 (20.3) of Heterophyes nocens, 6 (3.0) of Heterophyopsis continua, and 1 (1.0) of Stictodora fuscata. The results indicated that the surveyed area is a highly endemic area of metagonimiasis (three Metagonimus species) with low-grade mixed infections of 3 other heterophyid flukes. The infected people experienced variable degrees of gastrointestinal discomfort and indigestion. They consumed raw freshwater and brackish water fish, including sweetfish and mullets. It is strongly recommended that people residing in the survey area avoid eating raw fish to prevent M. yokogawai and other heterophyid infections.

Import of exotic and zoonotic trematodes (Heterophyidae: Centrocestus sp.) in Xiphophorus maculatus: implications for ornamental fish import control in Europe.

Ornamental fish, Xiphophorus maculatus, were imported from Singapore to Denmark for distribution to local aquarists. Importers observed lethargic and erratic swimming patterns among fish and forwarded a total of 30 fish for pathological examination to a university diagnostic service. All fish were diagnosed infected with encysted Centrocestus sp. metacercariae in gills (prevalence of 100% and mean intensity of 454.5 ± 161.9 parasites per fish). Metacercariae were identified by morphological and molecular methods. Cysts (mean length 163.3 ± 13.7 µm and mean width 113.3 ± 10.6 µm) contained a bent metacercaria with an X-shaped excretory bladder. PCR amplification of a rDNA region (5.8S rRNA gene, ITS-2, 28S rRNA gene) and subsequent sequencing confirmed the diagnosis. Metacercariae were found in gill filaments adjacent to the cartilage associated with cartilage hypertrophy, epithelial and mucous cell hyperplasia, clubbing and lamellar fusion. Host cell encapsulation of cysts comprised several layers of leucocytes, chondroblast-like and fibroblast-like cells. The observations raise concerns with regard to veterinary inspection and quarantine procedures. The zoonotic potential of these trematodes and a possible spread of the parasites in natural habitats in Europe should be regarded as a public health issue. So far, several cases of human infections have been reported only in Asia, but the potential intermediate host snail, Melanoides tuberculata, has been recorded in Germany. Accordingly, establishment of the parasite in Europe with climate changes should be considered a risk.

[A woman with long-lasting diarrhoea and fatigue]. / En ung kvinne med langvarig diaré og slapphet.

BACKGROUND: Intestinal trematodes are common parasites in man and many mammals. Infection is often asymptomatic and unrecognised. CASE PRESENTATION: A woman in her twenties presented with loose stools of variable intensity over six months. Additionally, she had experienced considerable fatigue during this period. There was no weight loss and initial blood tests were normal. Further testing at the second visit included stool microscopy, and small trematode eggs consistent with H. heterophyes infection were found. A more thorough anamnesis revealed the onset of symptoms on the day she returned from a week's holiday, and the probable exposure occurred from eating sushi twice during this holiday. After one day of treatment with praziquantel 40 mg/kg administered in three doses, the patient recovered completely within two to four weeks. Her asymptomatic partner had consumed the same food and had the same eggs in his stool sample. He was successfully treated with the same treatment dose. INTERPRETATION: A detailed travel history may provide important information relating to the diagnosis of diarrhoea and fatigue. Symptoms of H. heterophyes infection are variable. A single day's dose of 40 mg/kg of praziquantel was sufficient to eradicate infection in the two cases presented.

High prevalence of haplorchiasis in Nan and Lampang provinces, Thailand, proven by adult worm recovery from suspected opisthorchiasis cases.

Opisthorchiasis, a risk factor for cholangiocarcinoma in humans, is of public health importance in Thailand. The Annual Surveillance Reports from Nan and Lampang Provinces, Thailand, for the year 2011 showed an opisthorchiasis prevalence of over 70% by recovery of eggs in the feces. This study investigated whether most cases are actually due to minute intestinal flukes (MIF) rather than Opisthorchis viverrini, as the eggs of both can hardly be differentiated by morphology. Fifty and 100 cases from residents in Nan and Lampang, respectively, had stools positive for eggs initially assumed to be those of O. viverrini. Each patient was given praziquantel at 40 mg/kg in a single dose. After 2 hr, 30-45 ml of the purgative magnesium sulfate was given, and stools were collected up to 4 times sequentially. The stools were examined for adult worms by simple sedimentation. It was found that 39 of 50 cases (78.0%) from Nan Province had Haplorchis taichui, with intensities ranging from 5 to 1,250 with an average of 62 worms/case. Taenia saginata (7 cases) and Enterobius vermicularis (1 case) were other helminths recovered as the co-infectants. In Lampang Province, H. taichui was recovered from 69 cases (69.0%). The number of flukes recovered ranged from 1 to 4,277, with an average of 326 worms/case. Four cases had Phaneropsolus bonnei, and 10 T. saginata as the co-infectants. Adult specimens of O. viverrini were not recovered from any stool. Clearly, MIF infection, especially haplorchiasis, is more common in northern Thailand. These findings should encourage the Public Health Office to employ more specific tools than Kato's method for surveillance of opisthorchiasis in Thailand.

Copro-DNA diagnosis of Opisthorchis viverrini and Haplorchis taichui infection in an endemic area of Lao PDR.

The utility of differential copro-DNA diagnosis using modified sample preparation steps of small liver and minute intestinal fluke infections was tested. Fecal samples containing parasite eggs were washed extensively with diluted detergent solution. Parasite eggs were concentrated by sedimentation and broken by microwaving before DNA extraction. PCR targeting ITS1 and ITS2 regions were performed using primer specific for Opisthorchis viverrini, Haplorchis taichui and other related species. Of 125 fecal samples, 94 were positive for small trematode eggs by a modified cellophane thick smear method. By ITS1-PCR, 52 samples were positive for O. viverrini, 12 H. taichui and 7 mixed infection. By ITS2-PCR, 63 were positive for O. viverini, 17 H. taichui, and 19 mixed infection. The ITS-PCR assay identified a higher number of opisthorchiasis cases than those with O. viverrini expelled after treatment, but for H. taichui, ITS-PCR identified less than half of the worm expelled cases. These results showed that copro-DNA diagnosis was useful for the differential diagnosis of O. viverrini and H. taichui infection, which could not be discriminated by microscopy.

Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR.

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.

Case of metagonimiasis complicated with multiple intracerebral hemorrhages and diabetes mellitus.

Metagonimiasis yokogawai is an unusual intestinal parasitic disease caused by metacercariae of Metagonimus yokogawai. The first clinical manifestations of this disease do not always correlate with gastrointestinal signs. A 61-year-old man with left hemiparesis and disorientation was admitted to our hospital because of atypical nonhypertensive multiple intracerebral hemorrhages, which were conservatively treated. The patient was discharged from our hospital after 2 months without any neurological deficits; however, he was readmitted owing to a body temperature higher than 38 degrees C for nearly 2 weeks. Examination of stool revealed eggs of M. yokogawai, and the body temperature returned to normal after administration of praziquantel. Furthermore, the control of the patient's diabetes mellitus (DM) markedly improved after the treatment, although the patient had had DM for more than 2 years. We conclude that DM is a chronic sign of metagonimiasis in carriers and that intracerebral hemorrhage might be an acute sign in the aggravated phase of the disease.

A new endemic focus of Heterophyes nocens and other heterophyid infections in a coastal area of Gangjin-gun, Jeollanam-do.

To know the prevalence of heterophyid trematodes among inhabitants of a southern coastal village, i.e., Sacho-ri, Gangjin-gun, Jeollanam-do (Province), 82 stool samples were examined on helminth eggs and protozoan cysts using Kato-Katz and formalin-ether sedimentation techniques. Total 33 people (40.2%) were positive for trematodes (Heterophyes nocens 15 people, Pygidiopsis summa 3, Metagonimus sp.; 4, Clonorchis sinensis 7, Gymnophalloides seoi 6) and/or protozoa (Entamoeba coli 3). Among intestinal trematode egg positive cases, 17 were treated with praziquantel and their whole diarrheic stools were collected after purgation. Adult flukes of H. nocens (number of specimens = 1,294), P. summa (386), Stellantchasmus falcatus (5), Stictodora lari (4), and Heterophyopsis continua (1) were collected using a stereomicroscope. To know the source of human H. nocens infections in this village, metacercarial infections in mullets (10) were examined and most H. nocens metacercariae (101/105, 96.2%) were found in the trunk portion. From above results, the surveyed coastal village has been newly known as an endemic focus of human H. nocens infection and consuming raw mullets was the presumable source of human heterophyid infections.

Prevalence of Gymnophalloides seoi infection in coastal villages of Haenam-gun and Yeongam-gun, Republic of Korea.

One coastal village in Haenam-gun and two in Yeongam-gun, Jeollanam-do were surveyed for intestinal parasite infections by fecal examination. The egg positive rates of Gymnophalloides seoi were high, 24.1% (14/58) in Haenam-gun and 9.3% (11/118) in Yeongam-gun. The egg positive rates of heterophyids, including Heterophyes nocens, and of Clonorchis sinensis were 10.3% and 6.9% in Haenam-gun, and 14.4% and 8.5% in Yeongam-gun, respectively. After praziquantel treatment and purgation, a total of 37,761 fluke specimens were recovered from 17 patients; 11 in Haenam-gun and 6 in Yeongam-gun. Gymnophalloides seoi was the most commonly recovered species, with 37,489 specimens in total (2,205 per person). Other recovered flukes included Heterophyes nocens, Stictodora fuscata, Heterophyopsis continua, Pygidiopsis summa, and undetermined species. These results indicate that the areas surveyed are new endemic foci of G. seoi.

Melanoides tuberculatus (Gastropoda: Thiaridae) as intermediate host of Heterophyidae (Trematoda: Digenea) in Rio de Janeiro metropolitan area, Brazil

No final da década de 60, caramujos da espécie Melanoides tuberculatus, originários do nordeste africano e sudeste asiático, foram introduzidos no Brasil. Os primeiros registros de espécimes infectados com cercárias foram feitos no Estado do Rio de Janeiro em 2001. O presente estudo relata a ocorrência de M. tuberculatus infectados com larvas de trematódeos na cidade do Rio de Janeiro. O sedimento dos criadouros foi coletado e peneirado através de malhas de 0,25 polegadas. Os caramujos foram transportados em recipientes com água dos criadouros, sendo medidos e posteriormente individualizados. Eles foram expostos à luz e temperatura artificiais para induzir a emergência cercariana. As cercárias mais ativas foram processadas pelos métodos de coloração diferencial e de impregnação por nitrato de prata. Os caramujos negativos foram subsequentemente dissecados. Aproximadamente 700 caramujos foram coletados. Seu comprimento variou de 1,2 a 3,3 cm. A taxa de prevalência foi de 15,76% apesar de 53,76% dos caramujos estarem infectados em uma das áreas. Os caramujos estavam infectados com rédias e cercárias do tipo pleurolofocerca. A morfologia e a quetotaxia cercarianas foram compatíveis com as da família Heterophyidae principalmente devido à presença de nadadeiras medianas dorsais e ventrais e à ausência de receptores sensoriais CI dorsais.

Growth and development of Massaliatrema misgurni (Trematoda: Heterophyidae) in mice and its metacercarial morphology.

The morphology of metacercariae of Massaliatrema misgurni Ohyama et al. (Ohyama et al., Parasitol Int 2001; 50; 267-71) was described, and their infectivity, egg output, growth and development in mice until day 35 post infection (PI) were studied. Metacercarial cysts from loaches imported from China to Japan were 199-349 microm in diameter and consisted of a very thick translucent outer layer and a refractile inner layer. Excysted metacercariae basically had the shape of miniature adults, and a pair of pre-developed testes but no other genital organs were recognized. The worm recovery rate from mice was 36.7-51.7% during days 3-7 PI, and decreased remarkably to 2.5 and 1.7% at days 28 and 35 PI. The prepatent period was 3-4 days, and the egg output quickly increased and sustained high levels at days 5-7 PI, then decreased suddenly at day 8 PI, and continued at a low level until day 28 PI. The size of the body and inner organs such as the oral sucker, pharynx, acetabulum, testes, ovary and seminal receptacle quickly increased until day 3 PI, and sustained at a plateau level until day 21 PI except testes which gradually decreased until 21 PI. The number of the uterine eggs increased with a short time lag compared to other genital organs and sustained a plateau level until day 21 PI. Compared with other Heterophyidae species, M. misgurni was characterized by the remarkably fast growth and development.

Intestinal fluke infections: the heterophyids.

The food-borne small flukes infections are uncommon in Italy, because the parasite is strictly limited to certain areas of the world. Humans become infected by ingestion of parasitized fish that is raw, inadequately cooked or improperly-pickled or salted. Two case reports of intestinal infections by Heterophyies heterophyies are reported.

An incidental case of human Heterophyes nocens infection diagnosed by sectional morphology in a biopsy specimen of the small intestine.

A case of human infection with Heterophyes nocens (Heterophyidae) was incidentally found in a biopsy specimen of the Meckel's diverticulum at the upper part of the small intestine. The patient was a 58-year-old man living in a rural area of Talsonggun, Kyongsangbuk-do. He had gastrointestinal symptoms such as epigastric pain, indigestion, and abdominal discomfort for 3 months, and severe diarrhea, abdominal pain, and vomiting for about 1 month before hospitalization. Endoscopy of the upper part of the small intestine revealed a Meckel's diverticulum, and it was excised and histopathologically examined. Three adult flukes were incidentally found sectioned in the mucosa, and they were identified as H. nocens. The patient had a history of eating raw mullets at a fish market in Pusan 6 months ago, and the mullets were presumed to be the source of infection. This case brings a considerable interest in that specific diagnosis of heterophyid infections could be done by sectional morphology of the worms.

Human infection of Centrocestus caninus in Thailand.

Examination of fecal samples, collected after treatment with praziquantel, of the persons positive with opisthorchioid eggs residing in the northern provinces of Thailand, was carried out. Five adults Centrocestus were recovered in one sample from Doi Saket, Chiang Mai and one adult was found in another fecal sample from Wiang Pa Pao, Chiang Rai. The worms had 26-30 spines arranging in two rows around the oral sucker which agreed with the character of C. caninus. This is the first record of natural human infection with Centrocestus in Thailand.

Stictodora fuscatum (Heterophyidae) metacercariae encysted in gobies, Acanthogobius flavimanus.

The metacercariae of the genus Stictodora encysted in gobies, Acanthogobius flavimanus, purchased from a local market of Haenam-gun, Chollanam-do, were identified as those of S. fuscatum. A total of 1,437 metacercariae were collected from 30 gobies and some of them were orally fed to cats in order to obtain the adult worms. The metacercariae were 0.194-0.301 mm by 0.168-0.270 mm in size, yellowish brown, round to elliptical, and with a thin and transparent cyst wall. The excysted larvae were 0.280-0.423 mm long and 0.150-0.184 mm wide, beset with numerous tegumental spines on the anterior half of the body, and equipped with a characteristic ventrogenital sac. Total 21 adult worms were recovered from 2 cats 7 days after infection. The adult worms were small, leaf-like, 0.514-0.918 mm long and 0.235-0.377 mm wide. The ventrogenital sac was elliptical and its gonotyl was armed with 13-18 spines which were radially arranged and appeared as fan-shape. It has been confirmed that A. flavimanus is a second intermediate host of S. fuscatum in Korea.