Malariometric survey in Keoudom District, Laos: sensitivity of Plasmodium falciparum to anti-malarials and automedication with chloroquine.

A malariometric survey was conducted in the Keoudom District, in the northern part of Vientiane Province, Laos, where an artificial dam-lake on the Nam River is located. The parasite rate of the whole cohort representing 1,105 subjects was 2.44% with the predominance of Plasmodium vivax (70%), while P. falciparum represented 30% with the average parasite density index 3. The low spleen rate (2.3%) characterized the study area as a hypoendemic zone. IFAT antibodies were examined in 419 subjects. The seropositivity of 195 persons < 15 years was 13.7% while in > 15 year old subjects seropositivity was 61.6% with a low GMRT in both groups (140:148). Automedication with aminoquinoline was assayed by urinary analysis in 125 outpatients. Of these, 36 (28.8%) were positive, 89 (71.2%) negative. The frequency of positive blood films for P. falciparum was higher in subjects with aminoquinoline in the urine (36.1%) than in those without (10.1%). Chloroquine sensitivity assay of 15 strains of P. falciparum displayed resistance in 39.3%.

Current status of food-borne parasitic zoonoses in Laos.

Stool samples from a total of 1,008 persons were examined for intestinal parasites during a comprehensive study on the epidemiology of small fluke infections in Laos. The prevalence of small fluke eggs in the stool was seen to peak at age 20 years, particularly in men from villages (90.4%). Three quarters of infections belong to the category of light infections and only 0.6% to the category of very heavy according to eggs per gram of feces (EPG). The highest EPG was in the 11-15 year age group. In addition, the following parasites were diagnosed: Sarcocystis hominis (prevalence more than 10% in the group over 20 years of age), Taenia sp. (12.4% for the village people over 20 years), Fasciolopsts buski (3.8% for the same group). The habit of Laos people to eat raw fish, beef and pork flesh, is reflected in significant epidemiological consequences. Cercariae of Opisthorchis viverrini occurred in 0.5% of Bithynia siamensis goniomphalus examined, Haplorchis sp. cercariae were found in 0.9% of Tarebia granifera snails. Metacercariae of O. viverrini were found in flesh of 7 species of cyprinid fish. Haplorchis taichui in 4 species of these fish, and H. pumilio of two cyprinid species. Hampala macrolepidota harbored larvae of all above mentioned species. Stellantchasmus falcatus was recovered in fins of belonid fish Xenentodon cancila. Adults of O. viverrini were found in 36% of domestic and stray cats, Heterophyid flukes were found in 24% cats. The most frequent species were H. taichui and H. yokogawai. Eight Laotian students were treated in Czechoslovakia with praziquantel to determine fluke infection. Three were infected only by O. viverrini, four only by H. taichui and one by both O. viverrini and H. taichui. These results demonstrate the problem of correct differential diagnosis of food-borne small fluke infections and the need to assess the clinical course and public health aspects of infections.

Serological differentiation of human small fluke infections using Opisthorchis viverrini and Haplorchis taichui antigens.

Sera from 642 inhabitants of Vientiane Province (Laos) were examined by enzyme-linked immunosorbent assay (ELISA) using cytoplasmic and membranous antigens prepared from adult worms. Worms of Opisthorchis viverrini originated from liver of dissected cats, Haplorchis taichui were obtained from a stool specimen of a Laotian patient after praziquantel treatment. The sera were divided into five groups according to the intensity of infection expressed as egg count per gram of patients stool (EPG). Correlation between intensity of infection and the level of antibodies in serum was recorded. Reactions obtained using the cytoplasmic antigens were more sensitive and more specific compared to those with membranous antigens. Cross-reactions between antigens of both helminth species were found. Highly positive sera were examined using electroimmunotransfer blots (EITB) with cytoplasmic antigens of both species, which enabled the species differentiation. Antigens of both species yielded several shared fractions; however, differences between them were found: homologous sera reacted specifically with O. viverrini antigen in the area of 70 kDa and with H. taichui antigen in the area of 10 kDa. Thirty-one of 122 tested sera had specific antibodies against O. viverrini, 77 sera against H. taichui and 14 sera against both species. The results confirmed our assumption about predominant occurrence of heterophyid flukes in the human population living in studied area, compared with the occurrence of opisthorchid flukes. Hence, serology seems to be helpful tool for correct diagnosis of small fluke infections.

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.

Study of the body surface of Haplorchis yokogawai (Katsuta, 1932) and H. taichui (Nishigori, 1924) (Trematoda:Heterophyidae).

The morphology of the surface of two heterophyid flukes, Haplorchis yokogawai and H. taichui, that can infect humans eating raw fish flesh, has been studied using scanning and transmission electron microscopy. The body surface of both species is covered with scale-like, serrated spines with the exception of areas around the oral sucker, ventrogenital opening and excretory pore. The species slightly differ by the dentation pattern of tegumental spines: H. yokogawai has finer and more numerous spine teeth (maximum tooth number 14-16) while H. taichui has more serrated spines, mostly with a lower number (up to 10-12) of somewhat more robust teeth. However, because of the variability, the shape and dentation of spines do not seem to be reliable diagnostic features enabling mutual differentiation of both species.

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.