The anti-amoebic activity of some medicinal plants used by AIDS patients in southern Thailand.

The anti-amoebic activities of chloroform, methanol and water extracts from 12 Thai medicinal plants (39 extracts) commonly used by AIDS patients in southern Thailand were screened, at a concentration of 1,000 microg/ml, against Entamoeba histolytica strain HTH-56:MUTM and strain HM1:IMSS growing in vitro. The extracts were incubated with 2x10(5) E. histolytica trophozoites/ml of medium at 37 degrees C under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and scored (1-4) according to the appearance and numbers of the trophozoites. The extracts that caused inhibition were selected and retested using the same conditions but with concentrations that ranged from 31.25 to 1,000 microg/ml using E. histolytica strain HM1:IMSS, and the IC(50) values for each extract were calculated. The chloroform extracts from Alpinia galanga (IC(50) 55.2 microg/ml), Barleria lupulina (IC(50) 78.5 microg/ml), Boesenbergia pandurata (IC(50) 45.8 microg/ml), Piper betle (IC(50) 91.1 microg/ml) and Piper chaba (IC(50) 71.4 microg/ml) and the methanol extract from B. pandurata (IC(50) 57.6 microg/ml) were all classified as "active", i.e. with an IC(50) of less than 100 microg/ml, whereas those from Murraya paniculata (IC(50) 116.5 microg/ml) and Zingiber zerumbet (IC(50) 196.9 microg/ml) were classified as being "moderately active". The IC(50) of a standard drug, metronidazole, was 1.1 microg/ml.

Diagnosis of intestinal amebiasis using salivary IgA antibody detection.

Attempts were made to use soluble antigen extract of strain HK-9 of Entamoeba histolytica to detect salivary IgA antibodies in intestinal amebiasis patients by using ELISA. Total salivary samples of 109 individuals were divided into four groups. Group I comprised 32 patients whose stools were positive only for E. histolytica cysts and/or trophozoites. Group II comprised 12 individuals whose stools were positive for E. histolytica and other intestinal parasites. Group III comprised 36 individuals whose stools were negative for E. histolytica but contained other intestinal parasites such as E. coli, E. nana, Blastocystis hominis, Trichomonas hominis, Giardia lamblia, Opisthorchis viverrini, and hookworm. Group IV comprised 29 healthy individuals whose stools were free from any intestinal parasitic infections. Based on the mean optical density, OD + 2SD of the results from 29 parasitologically negative healthy individuals, the cut-off OD value for salivary IgA antibodies was 1.265. Therefore, the assays were positive in 14 out of 32 (43.75%) of group I and 2 out of 12 (16.6%) of group II. The assays were positive in 16 out of 36 (44.44%) for group III whereas 2 out of 29 (6.90%) for group IV were positive. The overall sensitivity and specificity of the assays were 36% and 72%, respectively. The false positive rate was 28% and the false negative rate was 64%. The predictive values of positive and negative results were 47% and 63%, respectively. The diagnostic accuracy of ELISA for the presence of salivary IgA antibodies was 58%.

Comparison between R-phycocyanin-labeled and R-phycoerythrin-labeled monoclonal antibody (Mab) probes for the detection of Entamoeba histolytica trophozoites.

A comparison between R-phycocyanin (R-PC)-labeled monoclonal antibody (MAb) probe and R-phycoerythrin (R-PE)-labeled MAb probe for the detection of the three standard reference strains of the cultured-derived Entamoeba histolytica trophozoites, namely HK-9, HM-1:IMSS, and HTH-56:MUTM were evaluated by using direct immunofluorescence antibody (DIFA) assay five times for each strain. Under the blue irradiation of the fluorescent microscope, both R-PC-labeled and R-PE-labeled MAb probes showed consistently greenish-yellow trophozoites and golden-orange trophozoites, respectively. The R-PE-labeled MAb probe stained the trophozoites more brightly and clearly than those stained by the R-PC-labeled MAb probe of the same Eh208C2-2MAb. When observed under the green irradiation, both probes showed the same intensity of brightly red color at the trophozoites of all three strains of E. histolytica. The sensitivity of both tests was 100%. Since this Eh208C2-2MAb could recognize specifically E. histolytica pyruvate:ferredoxin oxidoreductase (PFOR) enzyme, therefore, our two antibody probes would be valuable for use as a rapid, easy and sensitive test for diagnosis of invasive amebiasis. Further applications of these two probes directly onto the fecal sample spots and to more culture-derived strains of E. histolytica/E. dispar of known zymodemes in collaboration with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDRB), Dhaka, Bangladesh, are under investigation.

Monoclonal antibodies to quinine.

Monoclonal antibodies (MAbs) to quinine conjugated to a carrier protein were produced. Quinine was converted into a hemisuccinate prior to covalently linked to bovine serum albumin (BSA) by reacting with N,N'-disuccinimidyl carbonate (DSC). Coupling ratio of quinine-BSA was 13:1 calculated by spectrophotometry and 14:1 by calculation from quinine standard curve. This immunogen was used for both monoclonal antibody production and for screening test, indirect ELISA. The specificity of quinine-BSA MAbs was examined by checking the cross reactivity with BSA and the structurally related antimalarial drug, mefloquine. Six MAbs belonging to IgG1 were obtained. These MAbs slightly reacted with mefloquine-BSA because of closely related structure of mefloquine to quinine and similar conjugate preparation procedure used for conjugation. One selected MAb against quinine-BSA, showed higher reactivity with blood samples from patients previously treated with quinine when compared to normal blood. This preliminary test indicated that MAbs obtained may be useful to be used as the probe for detection of quinine in biological fluids.

Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica.

Metronidazole resistance has been induced in an axenic strain of Entamoeba histolytica (HTH-56:MUTM) following continuous exposure to steadily increasing drug concentrations. The drug-resistant line is routinely maintained in normally lethal levels of metronidazole (10 microM). Resistance to this concentration of drug was developed over 177 days. Decreased pyruvate:ferredoxin oxidoreductase (PFOR) activity in anaerobic organisms is one mechanism of metronidazole resistance but in entamoeba, PFOR activity was not decreased in metronidazole-resistant parasites as determined by immunofluorescent assays and immunoblotting studies. 2-Oxoacid oxidoreductase activity, which appeared to be due to a single enzyme, PFOR, was evident with pyruvate as well as the alternative substrates, alpha-ketobutyrate, alpha-ketoglutarate and oxaloacetate. A marked increase in superoxide dismutase (SOD) activity was detected in metronidazole-resistant E. histolytica. Increased SOD activity has not previously been documented as a mechanism of drug resistance although SOD has been associated with a range of stress situations in other organisms.

Monoclonal antibody-based ELISA for the detection of circulating Entamoeba histolytica antigen in hepatic amebiasis in hamsters (Mesocricetus auratus).

Circulating amebic antigens were determined by using a sandwich ELISA with specific monoclonal antibody in the sera of 35 group I hamsters, 7 of which were sacrificed at intervals after hepatic inoculation with 500,000 axenically grown HM:1:IMSS strain of E. histolytica trophozoites, 7 group II infected hamsters in which metronidazole treatment was given and 18 group III uninfected controls. Amebic antigenemia was demonstrated in 5 of 7 (71.4%), 6 of 7 (85.7%), 7 of 7 (100%), 7 of 7 (100%) and 7 of 7 (100%) of group I hamsters on days 5, 10, 15, 20 and 30 of infections respectively, whereas 6 of 7 (85.7%) of group II hamsters were weakly positive, one was negative and all 18 group III hamsters were negative. The sensitivity of the assay was 100% after the animals were infected 15 days onwards. The level of antigenemia in hamsters of group I with abscess was significantly higher than those of the same group without abscess (p < 0.05). Absence or reduction of antigenemia after treatment could be interpreted to mean a positive test of cure and favorable therapeutic response. The MAb-PAb-based ELISA for the detection of circulating E. histolytica represents a simple and sensitive diagnostic test for invasive amebiasis in hamsters. Application of this test in amebic liver abscess patients should be of diagnostic value for indication of present infection or test of cure after successful treatment.

Application of local products R-phycoerythrin and monoclonal antibody as a fluorescent antibody probe to detect Entamoeba histolytica trophozoites.

R-phycoerythrin (R-PE) was extracted from red algae, Gracilaria fisheri from Pattani Province, Thailand, with 50 mM sodium phosphate buffer, pH 7.0, followed by precipitation with 30-50% final concentration of saturated ammonium sulphate solution at 0 degree C. The precipitate was further purified by DEAE-cellulose (DE-52) column chromatography. The purified R-PE showed a single band of Mr 240 kDa by polyacrylamide gel electrophoresis with the maximum absorption and maximum fluorescence emission at 565 nm and at 573 nm respectively, and the OD ratio of 565 to 280 nm was 6.7. The IgG fraction of a murine monoclonal antibody (Eh208C2-2 MIgG) raised against trophozoites of HM-1: IMSS strain of pathogenic E. histolytica was conjugated with purified R-PE by using the heterobifunctional compound N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). The conjugate was shown by direct immunofluorescent antibody (DIFA) assay to stain specifically both the culture-derived and stool-derived E. histolytica trophozoites.

Pyruvate: ferredoxin oxidoreductase from Entamoeba histolytica recognized by a monoclonal antibody.

A mouse monoclonal antibody, Eh208C2-2 MAb, raised against whole cell antigens of Entamoeba histolytica trophozoites of the pathogenic strain HM-1: IMSS and polyclonal antisera (PAb) against membrane antigens of E. histolytica trophozoites of strain HTH-56: MUTM were screened against a cDNA library of the pathogenic strain, SFL3. The monoconal antibody detected many phage plaques expressing an E. histolytica protein. The DNA sequence encoding the protein was approximately 55% identical, over 1,100bp, to Trichomonas vaginalis pyruvate: ferredoxin oxidoreductase (PFOR) and pyruvate: flavodoxin oxidoreductase from Klebsiella pneumoniae, Anabaena variabilis and Enterobacter agglomerans. Two of seven clones detected by mouse polyclonal antisera also encoded this protein. Two others encoded Entamoeba Hsp70, another encoded Entamoeba alkyl-hydroperoxide reductase and the remaining two were unidentified sequences. Entamoeba PFOR is an abundant, antigenic protein which may be a useful target for the development of protective host immune responses against invasive amebiasis.

Comparison between microscopic examination, ELISA and quantitative buffy coat analysis in the diagnosis of falciparum malaria in an endemic population.

Monoclonal antibody-based ELISA and QBC (quantitative buffy coat analysis) were tested in two endemic areas with low and high incidence of malaria in Kanchanaburi Province, West Thailand with annual parasite incidence in 1992 of 119 and 5 per 1,000 population, respectively. The numbers of individuals positive by thick blood film examination (TBF) for P. falciparum with or without P. vivax, and P. vivax only were 82 and 69, respectively. The detection limit of ELISA was 10 parasites/10(6) red blood cells (RBC) (0.001% parasitemia). Of 1,095 individuals involved in the study at the beginning of the study, ELISA showed sensitivity, specificity, positive predictive value and negative predictive value of 78.1%, 94.9%, 72% and 98.1%, respectively. Nine of 18 (50%) TBF-positive but ELISA-positive individuals had parasitemia of less than 10 parasites/10(6) RBC. High and low incidence areas did not affect the validity of our result. Regression analysis showed good correlation between log parasitemia and ELISA percent OD increase (Y = 0 + 64.9*logX, r = 0.65), and agreement between TBF and ELISA results was 95.9%. In a fortnightly follow-up, in 82 TBF-positive individuals, both ELISA and TBF positive rates correlatively declined with agreement of 96.3%. With samples taken on the first day of the study, the TBF and QBC results were also correlated with agreement of 95.8% for P. falciparum, 95.6% for P. vivax. During 8 week follow-up involving altogether 191 samples, agreement between TBF and QBC results were 87.4% for P. falciparum. QBC detected more cases with P. falciparum infections but detected smaller number of cases with P. vivax infections.

Monoclonal antibody-based immunohistochemical demonstration of Entamoeba histolytica in liver tissues of experimentally infected hamster (Mesocricetus auratus).

Histopathological changes and the presence of Entamoeba histolytica trophozoites was sequentially followed after intrahepatic inoculation of the parasites in 42 hamsters, 35 of which received no treatment whereas the remaining seven were treated with metronidazole. The liver tissues were examined for amoebic trophozoites by a monoclonal antibody (mAb)-based immunofluorescence assay (IFA), a mAb-based immunoperoxidase (IPx) and H & E staining. The number of hamsters developing abscesses was increased with time and was highest on day 30. Cellular infiltration with inflammatory cells and glycogen depletion were observed as early as day 5, followed thereafter by more intense inflammation of portal canals, periportal fibrosis, bile duct proliferation and hepatocyte degeneration. In 7 metronidazole-treated hamsters, no obvious pathological damage was seen. In a group of seven hamsters each, both IPx and IFA were positive in 3, 3, 4, 5 and 4 hamsters and in 3, 4, 3, 3 and 5 hamsters on days 5, 10, 15, 20 and 30, respectively. In 18 control hamsters, IPx, IFA and H & E were all negative. If the result from H & E was used as a gold standard, agreement between H & E and IFA and H & E and IPx were 91.4%, and 88.6%, respectively. Sensitivity and specificity were 93.8% and 89.5%, respectively for IFA, and 93.8% and 84.2%, respectively for IPx.

Comparative horizontal starch gel isoenzyme electrophoresis of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Foot muscle tissue extracts from six lymnaeid species of the Indo-Pacific region [Lymnaea (Bullastra) cumingiana and L. (Radix) quadrasi from the Philippines, L. (R.) rubiginosa from Indonesia and Thailand, and L. (R.) viridis from Guam and Hong Kong] were subjected to horizontal starch gel isoenzyme electrophoresis and assayed for seven isoenzymes (AcP, AlP, CA, EST, LAP, CAT and GOT) to elucidate their taxonomic relationships. L. cumingiana exhibited banding patterns for EST, LAP and CAT uniquely different from the rest, thus supporting the hypothesis that it is a distinct species. Zymogram patterns for AlP, CA, EST and LAP attest to the close affinity between L. quadrasi and L. rubiginosa (Indonesia and Thailand). Minor differences suggest a closer relationship between the two geographical strains of L. rubiginosa than with L. quadrasi, lending support to the hypothesis that L. quadrasi is inseparable as a race or variety from the typical L. swinhoei Adams, which in turn is but a race of L. auricularia, which also encompasses L. rubiginosa. The two geographical strains of L. viridis from Guam and Hong Kong showed the greatest consistency with regards to similarity and congruence in banding patterns. Non-specific esterases (EST) were the most useful in distinguishing the six species from each other.

Comparative radular morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

The radular morphology of Lymnaea (Bullastra) cumingiana was compared to that of five other Indo-Pacific lymnaeid "species", namely: L. (Radix) quadrasi (Philippines), L. (R.) rubiginosa (Indonesia and Thailand) and L. (R.) viridix (Guam and Hong Kong) in order to investigate the taxonomic relationship among the six species. Although all six species uniformly exhibited a unicuspid, slightly asymmetrical central (rachidian) tooth and tricuspid laterals, interesting differences were noted among the outer marginals. These were observed to be uniquely bicuspid in L. cumingiana, predominantly tricuspid in L. quadrasi, tetracuspid in L. rubiginosa (Indonesia and Thailand) and multicuspid in L. viridis (Guam and Hong Kong). Thus, the results support the hypotheses that L. cumingiana is a unique species compared to the rest, that L. quadrasi is closely related to L. rubiginosa (Indonesia and Thailand) and that the two geographical isolates of L. viridis have not diverged. Radular morphology was therefore found to have a limited significance in elucidating the taxonomic relationship between the six groups of lymnaeids studied.

Comparative shell morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Comparative shell morphology using both quantitative and qualitative parameters was employed to investigate the taxonomic relationship between the endemic Philippine species, Lymnaea (Bullastra) cumingiana and five other lymnaeid "species" in the Indo-Pacific region, namely: L. (Radix) quadrasi (Philippines). L. (Radix) rubiginosa (Indonesia), L. (Radix) rubiginosa (Thailand), L. (Radix) viridis (Guam) and L. (Radix) viridis (Hong Kong). Fifty randomly chosen adult specimens of each species were studied and compared, although only field-collected specimens were studied for the first four groups and laboratory-raised specimens for the last two group. Results strongly suggested that L. cumingiana is a distinct species among the rest. L. quadrasi, L. rubiginosa (Indonesia) and L. rubiginosa (Thailand) exhibited great affinity towards each other. Likewise, the two geographical isolates of L. viridis were practically identical to each other except for some minor size differences.

Ecological observations on Lymnaea (Bullastra) cumingiana.

Field surveys conducted at Echague, Isabela and San Pablo, Laguna revealed that Lymnaea (Bullastra) cumingiana, the natural second snail intermediate host of Echinostoma malayanum in the Philippines, exhibits a moderate degree of diversity in its choice of habitats. Rice fields of all stages of development, stagnant shallow streams and springs are the main areas where the snail can be collected from at Echague, Isabela. However, they were absent in rice fields that had been extensively sprayed with molluscicides to control the "golden apple snail" (Ampullarius canaliculatus). In contrast, they were also very abundant in the highly eutrophic waters of Sampaloc lake, San Pablo, Laguna. L. cumingiana co-exists with various species of insects, snails, fish and plants in these habitats. Information on ecological characteristics affecting its distribution will be useful for those who wish to collect and study this species in the future.

Axenic cultivation of Entamoeba histolytica from liver abscess and its zymodeme.

A local strain of Entamoeba histolytica, the HTH-56: MUTM from a human liver abscess was successfully axenized. The culture was initially established monoxenically in Diamond's TYI-S-33 medium in the presence of Crithidia luciliae and maintained at 34 +/- 0.5 degrees C. After 5 passages it was adapted to axenic cultivation by addition of 0.02% Bacto agar in Diamond's TYI-S-33 medium in place of Crithidia. Subcultures or replacement with fresh complete media were done twice or thrice for 7 days, after which the agar was omitted and a stable culture was obtained. Isoenzyme analysis showed that this strain of E. histolytica belonged to the zymodeme II pattern, which is one out of 10 pathogenic zymodemes of E. histolytica most commonly found among the virulent strains.

Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens.

A murine monoclonal antibody (MAb) (Eh208C2-2) raised against crude lysate of the pathogenic HM-1:IMSS strain of Entamoeba histolytica was used in an enzyme-linked immunosorbent assay for the detection of E. histolytica antigens in faecal specimens. The detection limit of the assay was 110 and 280 amoebae/ml of the HM-1:IMSS and HK-9 strains in phosphate-buffered saline, respectively. The assay was applied to single stool samples from 3 groups of individuals comprising 40 patients whose stools were positive for E. histolytica trophozoites and/or cysts (group I), 48 patients whose stools were negative for E. histolytica but positive for other parasites (group II), and 36 parasitologically-negative healthy controls (group III). Positivity rates of 77.5%, 2.1% and 2.7% were found in samples from groups I, II and III respectively. Specificity, positive and negative predictive values, and efficiency of the assay were 97.6%, 93.9%, 90.1% and 91.1% respectively. When group I samples were further divided into a trophozoite-positive subgroup IA (13 samples) and a cyst-positive subgroup IB (27 samples), the positive rates were 100% and 66.7%, respectively (P < 0.025).

Detection of shared antigens of human liver flukes Opisthorchis viverrini and its snail host, Bithynia spp.

The relationship between Opisthorchis viverrini and its snail intermediate host including Bithynia funiculata, B. siamensis siamensis and B. siamensis goniomphalos was carried out on the correlation of shared antigens and infection rates. B. funiculata and B. s. siamensis were equally susceptible to O. viverrini with relatively high infection rates of 72.2% and 69.9% respectively whereas B. s. goniomphalos gave lowest percentage of infection of only 9.6%. By immunoelectrophoresis, crude extract of O. viverrini adult worm produced the same number of two common precipitin bands of four different electrophoretic mobilities against anti-B. funiculata and anti-B. s. siamensis sera and one precipitin band in the antigen well region against anti-B. s. goniomphalos serum. The three snail antigens produced the same number of two common precipitin bands against anti-O. viverrini serum.

Competitive antibody binding inhibition ELISA for the detection of Plasmodium falciparum antigen.

A competitive antibody binding inhibition ELISA to detect Plasmodium falciparum-infected cells in clinical specimens was developed. Optimum conditions developed included: 12.5 micrograms/ml of P. falciparum antigen for plate coating, 25 micrograms/ml of polyclonal rabbit anti-P. falciparum IgG, 30 minute incubation of a mixture of infected red blood cell extract with anti-P. falciparum IgG, dilution of 1:500 of alkaline phosphatase-conjugated anti-rabbit IgG, and reading of the absorbance values 60 min after adding the p-nitrophenyl phosphate substrate. Reproducibility of the assay against cultured P. falciparum-infected red blood cells varied according to parasitemia, the higher the parasitemia, the better the reproducibility. The sensitivity of the assay was approximately 110 parasites/10(6) red blood cells. The assay was applied to field conditions involving 103 cases with falciparum malaria, 38 cases with vivax malaria and 30 healthy controls. With the 10% antibody binding inhibition as a cutoff, 87.4% of falciparum cases and 26.3% of vivax cases were positive. After treatment, the majority of cases became parasitologically negative with the corresponding negative assay. Regression analysis showed only weak but statistically significant correlation between the percent inhibition with parasitemia (r = 0.38, p less than 0.001), and this was more clearly shown in patients with high parasitemia.

Assessment of putative tests for protective immunity to falciparum malaria.

Sera from clinically immune individuals comprising 10 hospitalised patients (Group I), 30 persons residing in a malaria endemic area in Thailand (Group II) and 8 persons from a hyperendemic area in Ivory Coast (Group III) were tested by the parasite growth inhibition (PGI), indirect fluorescent antibody test of ring-infected erythrocyte surface antigen (RESA-IFA), urease-ELISA and Western blot. Paired sera from patients recovering from malaria (Group IV) as well as sera from blood donors were also tested. In the PGI test, sera were tested against three uncloned isolates of P. falciparum comprising SO, I4 and AE9 (PGI-SO, PGI-I4 and PGI-AE9 respectively). When growth inhibition of greater than or equal to 30% against any one of the three isolates was considered positive, the positive rate for the combined Groups I, II and III was 78.7%. Further analysis showed that the positive rates for PGI-SO, PGI-I4 and PGI-AE9 were 63.8%, 59.5% and 59.5% respectively and were not significantly different (p greater than 0.05). Comparison between PGI-SO, PGI-I4 and PGI-AE9 activities of Groups I, II and III sera showed no significant differences in any comparison groups except with PGI-AE9 in which Group III sera were more frequently positive than Group II sera (p = 0.004). Follow-up of PGI-SO and PGI-AE9 activities in Group IV patients showed mostly a decrease or no change in the activities of the convalescent sera taken 63 days later. RESA-IFA positive rate in the combined Groups I, II and III sera was 91.7%. There were no significant differences either in the seropositive rates or in the geometric mean antibody titers (GMT) between Groups I, II and III sera. Follow-up in Group IV patients showed no change in antibody titers in 64% of cases, decrease and increase in titers in 29% and 7% of cases respectively. The urease-ELISA seropositive rate in the combined Groups (I, II and III) was 89.5% which is not significantly different from that of RESA-IFA (p greater than 0.05). Comparison between individual Groups (I, II and III) likewise showed no significant differences in both GMT and seropositive rates. Follow-up in Group IV sera showed either no change or a decrease in antibody titers in 55.6% and 44.4% of cases respectively.(ABSTRACT TRUNCATED AT 400 WORDS)