Viroj Kitikoon

Identification and differentiation of human schistosomes by polymerase chain reaction.

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.

Specific monoclonal antibodies to Opisthorchis viverrini

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.

Possible protective immunity in human opisthorchiasis

Chronic infections with the liver flukes Opisthorchis viverrini and Clonorchis sinensis affect over 30 million people in southeastern Asia. With ongoing exposure, reinfection readily occurs following curative treatment and cumulative infections result in significant morbidity and a predisposition to cholangiocarcinoma. Though protective immunity has never been described in human opisthorchiasis, heterogeneity in worm burden occurs and a small number of exposed residents of endemic areas remain apparently uninfected. To explore the nature of this heterogeneity, we compared levels of serum antibody (Ab) to O. viverrini measured by an enzyme‐linked immunosor‐bent assay in 83 stool egg‐positive and 49 stool egg‐negative residents of an O. viverrini‐endemic area in Thailand. Compared to the egg‐positive residents, the egg‐negative group had significantly higher levels of immunoglobulin (Ig)G, IgA and IgM to adult worm homogenate (AWH) and total Ab to metacercaria homogenate (MH). Furthermore, immunoblot analyses revealed that a significantly higher proportion of sera from the egg‐negative residents had IgA reactivity against a 38‐k Da AWH antigen and IgM reactivity against carbohydrate epitopes of a 42‐k Da AWH glycoprotein antigen. These findings support a hypothesis that the egg‐negative group includes individuals who may be immunologically resistant to this usually chronic infection.

Field survey focused on Opisthorchis viverrini infection in five provinces of Cambodia.

BACKGROUND Opisthorchiasis is endemic in Thailand and Lao Peoples Democratic Republic and constitutes a major public health problem throughout the Mekong Basin. Although Cambodia is located in the Mekong Basin, the status of O. viverrini infection in that country was not previously clarified. This research was conducted to document the extent and distribution of O. viverrini infection in Cambodia. METHODS Surveillance was conducted in 55 villages in five Cambodian provinces. Research tools included stool examination using the Kato-Katz thick-smear technique, identification of intermediate hosts, and interviews covering factors related to O. viverrini infection. Some larvae and egg-positive stool samples were examined using PCR to detect O. viverrini DNA. RESULTS A total of 16,082 stool samples from the 55 villages were examined, of which 1232 were egg positive. In 15 villages with egg-positive rates of greater than 10%, eggs were found in 998 of 3585 stool samples, for an egg-positive rate of 27.8%. PCR analysis showed that 30 of 33 samples were positive for O. viverrini DNA from five villages in Kampong Cham and Kampong Thom provinces. The first intermediate host Bithynia siamensis siamensis was identified in the target areas of Takaev, Kandal, and Kampong Cham provinces. Cercariae were identified morphologically as O. viverrini and some were confirmed using PCR. Metacercariae of O. viverrini were identified by morphologic observations, animal experiments, or PCR in six species of fish in the target areas. DISCUSSION AND CONCLUSIONS Four Cambodian provinces were identified as endemic areas of O. viverrini infection. Careful planning is necessary for effective field surveys, because complex environmental factors might be involved in the distribution of O. viverrini infection-endemic areas in Cambodia. Many problems remain to be resolved regarding the status of O. viverrini infection in Cambodia, and a nationwide baseline survey is necessary.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.

Serum antibody responses in opisthorchiasis.

We evaluated an enzyme-linked immunosorbent assay using crude parasite homogenates as a diagnostic test for Opisthorchis viverrini infection in humans. Serum antibody (Ab) responses to O. viverrini adult worm homogenate (AWH) and metacercaria homogenate (MH) were studied in 83 infected residents of an opisthorchiasis-endemic area in Thailand. Elevated levels of Ab persisted for over 1 year following curative treatment with praziquantel, and cross-reactivity to O. viverrini AWH and MH antigens was observed in sera from individuals with other parasitic infections. Serum Ab to crude AWH and MH are therefore unsuitable for immunodiagnosis since they may be non-specific and would not differentiate between ongoing and past infection.

Serodiagnosis of human opisthorchiasis using cocktail and electroeluted Bithynia snail antigens.

Cocktail and electroeluted antigens from Bithynia goniomphalos, the snail intermediate host of Opisthorchis viverrini, were extracted and purified. The performance of these two antigens in the antibody detection of human opisthorchiasis was evaluated by indirect ELISA. Serum samples from people whose stool was either: (i). positive for Opisthorchis eggs (n=61); or (ii). positive for at least one of 19 other species of parasite (n=125); or (iii). clear of parasites (n=30) were tested. The sensitivity, specificity, positive predictive value and negative predictive value of ELISA using cocktail antigen were 88.5, 88, 78.2 and 94%, respectively; those of ELISA using eluted antigen (53 kDa) were 91.8, 98.4, 96.5 and 96.1%, respectively. Cross-reaction with the eluted antigen was seen in only one of four cases of hymenolepiasis and only one of 10 cases of strongyloidiasis. The kappa coefficients for ELISA in relation to stool examination were 0.84 (cocktail antigen) and 0.87 (eluted antigen). This study showed that Bithynia snail antigen could be used to replace worm antigen in the antibody detection of human O. viverrini infection.

Effect of Quail Litter Biochar on Productivity of Four New Physic Nut Varieties Planted in Cadmium-Contaminated Soil

Se ha visto que el biocarbon mejora la estructura del suelo y la retencion de agua, mejora la disponibilidad y la retencion de nutrientes, controla la acidez y reduce la toxicidad de metales pesados en las raices de las plantas. En este trabajo se investiga el uso de biocarbon de cama de codorniz (QLB) en la disponibilidad de Cd para la planta de jatrofa (Jatropha curcas L.) en un estudio de laboratorio. Se realiza una combinacion factorial con cuatro variedades nuevas de jatrofa (Takfa, Doi Saket, Lao y Rayong) sobre cuatro proporciones de QLB a 0, 5, 10, y 15 g kg-1 anadidos por separado a suelo contaminado con 60,8 mg Cd kg-1. Tras el trasplante se midio la altura de la planta y la cubierta vegetal cada 2 meses, el numero de hojas y ramas a los 6 meses y los parametros de rendimiento asi como el residuo de Cd a los 8 meses. A continuacion, tras la cosecha de la planta, se analizaron las caracteristicas quimicas, nutrientes y residuo de Cd en el suelo contaminado. El uso de QLB causo un aumento significativo en el potencial de crecimiento y en los parametros de rendimiento (P < 0,05), asi como una disminucion significativa del residuo de cadmio en las plantas (P < 0,05) y una mejora significativa en las caracteristicas quimicas, nivel de nutrientes y residuo de Cd en el suelo (P < 0,05). Se concluye que el uso de QLB puede disminuir significativamente la biodisponibilidad de Cd para la jatrofa, incrementar su potencial de crecimiento y rendimiento, y tiene el potencial de remediar el suelo contaminado con Cd. No obstante, no se aconseja el uso de QLB por encima de 15 g kg-1 de suelo. Dado que el QLB es de naturaleza alcalina puede afectar al pH del suelo.

Mekong schistosomiasis. III. A parasitological survey of domestic water buffalo (Bubalus bubalis) on Khong Island, Laos.

Of 103 water buffalo (Bubalus bubalis) examined on Khong Island by means of the M.I.F.C. and hatching techniques, none were passing eggs resimbling those of the Mekong schistosome. One buffalo calf was infected with Orientobilharzia harinasutai and another with Schistosoma spindale; this is the first time these parasites have been reported from Laos. Since the buffalo that were examined had constant and year-round access to a part of the Mekong River that has been shown to be a site of heavy transmission of schistosomiasis to humans and dogs, it was considered that the buffalo would have acquired the infection with the human Mekong schistosome if this were possible. In the absence of buffalo necropsies, and since no eggs of the Mekong schistosome were detected in the stools of these animals, we assumed that they had either not become infected with this parasite or, if they had, that the infections did not produce eggs in the faeces which were detectable by the methods employed. On the basis of our examinations, it would not seem that domestic water buffalo are involved as reservoirs in the epidemiology of human schistosomiasis on Khong Island.

Separation and characterization of adult worm proteins and glycoproteins from the liver fluke Opisthorchis viverrini.

Detailed studies of liver fluke proteins and antigens are necessary to facilitate further investigation of the human immune responses to these parasites. Accordingly, Opisthorchis viverrini antigens were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. We initially encountered excessive background smearing, vertical streaking, and indistinct bands that were similar to problems previously described by investigators studying this and other trematodes including Schistosoma mansoni. These problems were especially evident with silver staining of proteins and occurred despite the extensive use of protease inhibitors. They were minimized by using mini (vs. large) SDS-PAGE and Coomassie blue protein staining. With the latter 2 techniques, adult worm somatic proteins and excretory-secretory products were separated and characterized. Immunoblots using rabbit anti-adult worm sera demonstrated that some of these proteins were antigens common to both the adult and metacercarial stages. Several of these antigens also corresponded (according to molecular weight) to glycoproteins, detected by concanavalin A blotting. These findings form a base for subsequent studies of the human immune response to liver fluke infection.