Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans

BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

Some factors affecting Stellantchasmus falcatus metacercaria in laboratory.

Killing factors, various temperatures and solutions were studied in the laboratory on Stellantchasmus falcatus metacercaria in half-beaked fish (Dermogenus pusillus). Killing criteria followed the Movability Index from 1.000 within 24 hours. The metacercariae were collected from Chiang Mai moat. They were incubated in 0.85 % NaCl at -20 degrees C, room temperature, 4 degrees, 37 degrees, and 65 degrees C. The in vitro investigation showed that at -20 degrees C and 65 degrees C, the worms were killed within 18 and 2 hours, respectively, while other temperatures produced no effect. The solutions investigated were NaCl (10, 20, 30, and 40%), lemon juice (25, 50, 75, and 100%), acetic acid (5, 10, 20, and 30%), vinegar (1, 3, and 5%) and water as a control. The worms were killed in NaCl at 20, 30, and 40% within 12, 6, and 2 hours, respectively. Acetic acid at 5% and 10% killed the metacercaria within 12 and 6 hours while at 20% and 30%, within 2 hours. The killing effect of 3% vinegar was found within 18 hours and of 5% vinegar within 12 hours. Lemon juice showed no killing effect.

Investigation of Stellantchasmus falcatus metacercariae in half-beaked fish, Dermogenus pusillus from four districts of Chiang Mai Province, Thailand.

Half-beaked fish, Dermogenus pusillus, collected from Mueang, Hang Dong, Doi Saket and Saraphi Districts, Chiang Mai Province, were examined for their infection status with Stellantchasmus falcatus (Digenea: Heterophyidae) metacercariae. The infection rate of the fish was 100%. Fish in three of four districts were found only to have metacercariae of S. falcatus, whereas those in Saraphi District had mixed infections with metacercariae of Posthodiplostomum sp. The intensity of S. falcatus infection per fish varied; 652-1,342 (mean 999.5), 562-2,422 (1,323.1), 185-2,492 (502.6), and 22-550 (210.4) in Mueang, Hang Dong, Saraphi, and Doi Saket Districts, respectively. The body portions of the fish with the heaviest metacercarial infection were the muscles, in all districts. The present study confirms that half-beaked fish in Chiang Mai Province are heavily infected with S. falcatus metacercariae.

Trematodes of the family Opisthorchiidae: a minireview

Examination of the original descriptions of genera placed within the family Opisthorchiidae has revealed that only 33 of the original 43 genera are valid members of this family. Further study of these descriptions should also reveal that many of the subfamilies are also invalid. With reference to the original descriptions of these genera, and subsequent literature, a brief survey of the family has been compiled. Information on the spectrum of definitive hosts that these helminths parasitize is provided, as well as information on the life cycles, geographic distribution, and socioeconomic impacts of the family. More in-depth information is given on those species that are of particular medical importance; namely, Clonorchis sinensis, Opisthorchis viverrini, and O. felineus. The final aims of this review are to provide information on the entire genera of the family Opisthorchiidae, which will aid understanding of the phylogenetic relationships not only within the family, but also within the Class Trematoda.

Amphimerus bragai n.sp. (Digenea: Opisthorchiidae), a parasite of the rodent Nectomys squamipes (Cricetidae) from Minas Gerais, Brazil

Amphimerus bragai n.sp. (Digenea, Opisthorchiidae) from the bile ducts of a rodent from the State of Minas Gerais, Brazil, Nectomys squamipes (Cricetidae), is described. The new species was studied by both light and scanning electron microscopy. A table is presented comparing the measurements of the new species with those of A. lancea (Diesing, 1850) and A. vallecaucensis Thatcher, 1970, parasites of dolphins and marsupials, respectively. The new species is similar in size and body form to a A. vallecaucensis from which it differs in having a vitellarium that extends to the acetabulum while that of the former species are limited to the posterior one-third of the body. Additionally, the new species is from a rodent.

Metacercariae isolated from cyprinoid fishes in Khon Kaen District by digestion technic.

Several kinds of fresh-water fishes collected from natural water resources in Khon Kaen District were digested with 0.25% pepsin A to obtain the infective stage or metacercariae of fish-borne trematodes. Most of them harbored at least 3 species of trematodes. Two families of medical trematodes, i.e., Family heterophyidae and Family Opisthorchiidae, were isolated. The heterophyid flukes consisted of Haplorchis taichui, H. pumilio and Centrocestus formosanus. Opisthorchis viverrini was the only parasite of the Family Opisthorchiidae found in these fish. Among these four species, H. taichui was predominant and found in all kinds of fish examined, especially Hampala dispar.

Metacercariae in fishes of Sun Moon lake which is an endemic area for Clonorchis sinensis in Taiwan.

The Sun Moon lake in Central Taiwan is a known endemic area for clonorchiasis. Of the 45 fresh water fish, Hemiculter leucisculus, from the lake that were examined by artificial gastric juice digestion in October 1995, all were found to harbor metacercariae in their muscle. The number of metacercariae isolated from each fish ranged from 2 to 2,185, with an average of 254. A total of 11,443 metacercariae was collected from the 45 fish. Of the 4,223 metacercaria that were examined under light microscope, 4,064 (96.23%) were found to belong to Haplorchis taichui, 90(2.13%) to H. pumilio, 2(0.05%) to C. sinensis and 67 (1.59%) to unknown species due to the metacercariae being not yet developed or immature. The 2 C. sinensis metacercariae were obtained from 2 out of 45 fish examined. Our results contrast with reports of a decade ago which stated that all the fish of the Sun Moon lake examined were positive for C. sinensis. Possible reasons for the decrease of C. sinensis metacercariae are the disappearance of pig farms around the lake, increased awareness of the trematode by the lakeside inhabitants and probably the exclusive use of mammals as its definitive host by C. sinensis. In contrast, besides mammals, Haplorchis spp also use birds as their definitive hosts.

Differential diagnosis of opisthorchiid and heterophyid metacercariae (Trematoda) infecting flesh of cyprinid fish from Nam Ngum Dam Lake in Laos.

The examination of flesh of 313 cyprinid fish from the Nam Ngum water reservoir in Vientiane Province, Laos, revealed the presence of four morphologically similar opisthorchiid and heterophyid metacercariae of medical importance. The following morphological characters, visible under dissecting microscope with low magnification, were typical of trematodes recorded in fish: (1) Opisthorchis viverrini, the liver fluke, has oval cysts containing quickly moving C-shaped metacercariae provided with a rather large excretory bladder; (2) metacercariae of Haplorchis pumilio and H. taichui are surrounded by a thin-walled, oval cyst; they are actively motile, C- or S-shaped, and possess a relatively small excretory bladder; (3) cysts of Haplorchoides mehrai are considerably variable in shape, size, and thickness of cyst wall; larvae are slowly moving, C- or S-shaped, or often not folded, and provided with a rather small excretory bladder. Some cysts with dead or degenerated H. mehrai larvae are filled with brownish or yellowish pigment. Morphological variability of some larvae, however, excluded correct identification of all metacercariae during routine examination of fish. Therefore, we consider isolation of several larvae from each fish host as well as all "atypical" metacercariae from fish flesh and their examination under the light microscope to be necessary. Even though this method of examination is rather time consuming, it decreases the possibility of false identification of trematode larvae encysted in flesh of cyprinid fish during field studies on opisthorchosis and heterophyiosis.

Occurrence of some medically important flukes (Trematoda: Opisthorchiidae and Heterophyidae) in Nam Ngum water reservoir, Laos.

Occurrence of some medically important flukes transmitted to man by eating of raw fish was observed at 18 localities of Nam Ngum water reservoir, an important source of fish for inhabitants of Vientiane Province, Laos. Cercariae of Opisthorchis viverrini were recorded in 0.9% of Bithynia siamensis goniomphalus examined but in the most populated sites, where the density of the snails was the highest up to 2.2% of the intermediate hosts were infected. Haplorchis cercariae were found in 2.0% of Tarebia granifera examined. O. viverrini metacercariae were found in flesh of four fish species (Hampala dispar. H. macrolepidota, Puntius gonionotus, Cyclocheilichthys repasson) from 9 localities with the highest prevalence (16%) in H. dispar. Metacercariae of Haplorchis pumilio and H. taichui were found in flesh of Hampala dispar, H. macrolepidota and Puntius gonionotus with the prevalence of infection 2-6%. Metacercarie of Stellantchasmus falcatus were localized in fins of Xenentodon canciloides. Domestic cats (Felis catus f. domestica) harbored the following trematode species: O. viverrini, H. pumilio, H. taichui, H. yokogawai, S. falcatus. Eggs of small flukes were found in stools of 44% of examined inhabitants of 3 villages and of the district center Thinkeo. The study revealed that the best predictor of the existence of small fluke foci and their species composition in the area studied was the second intermediate host-fish.