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.

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%.

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.

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.

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 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.

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.

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.

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.

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)

Detection of antibodies against Opisthorchis viverrini in patients before and after treatment with praziquantel.

Levels of antibody in sera of 78 patients with opisthorchiasis, 30 patients with other liver diseases, 10 patients with schistosomiasis and 30 healthy individuals were compared using three serodiagnostic tests, namely indirect haemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA) and lectin immuno test (LIT). The geometric mean reciprocal titer in sera of opisthorchiasis patients was significantly higher than patients with other diseases, patients with schistosomiasis and healthy individuals (p less than 0.00001). After treatment with praziquantel, the antibody titers were decreased and became lowest 120 days after treatment. A statistically significant decrease from the pre-treatment sample was observed only at 120 days after infection and not earlier and only with ELISA (p = 0.03) and not with IHA and LIT (p greater than 0.05). Even with ELISA, significant decrease in antibody titer was apparent only when the pre-treatment sera had high enough antibody titer. ELISA was therefore better than the other two tests for the assessment of cure provided that the titer of pre-treatment sera was high.

Immunoprecipitating antibodies against blood stage antigens of Plasmodium falciparum in cerebral and non-cerebral malaria patients.

Immunoprecipitating antibodies were determined in paired sera of 31 patients with cerebral malaria (CM), of whom 14 had complicated cerebral malaria (CCM) and 17 had uncomplicated cerebral malaria (UCCM), 15 single specimens of patients with acute uncomplicated (AM) malaria taken on the day of admission and 8 healthy controls. All but one patient were admitted within the first three days of the onset of fever. More than 20 precipitating bands were observed, of which the predominating molecules were the Mr greater than 200, 180, 157, 135, 130, 115, 103, 96, 91, 73, 71, 61, 49, 45, 43, 41 and 14.3 Kd. In general, there were no significant differences in the positive rates among the AM, CCM and UCCM patients except for the pf135 Kd molecule which was more frequently reactive in UCCM patients than the AM and CCM patients. If immunological naiveness in term of protective immunity is the feature in CM patients, the immunoprecipitation test used is inadequate to demonstrate the fundamental differences in immune responses between CM and AM patients.

A comparative study of four methods for detecting antibody in asymptomatic giardiasis.

Four immunological methods for diagnosis of giardiasis comprising complement fixation (CF) test, indirect hemagglutination (IHA) test, enzyme-linked immuno sorbent assay (ELISA) and lectin immuno test (LIT) were studied. Fifty sera from asymptomatic giardiasis patients, 40 from patients with other diseases and 50 from healthy controls were evaluated. The seropositive rates in asymptomatic giardiasis were 36% for CF, 58% for LIT, 30% for IHA and 72% for ELISA. The seropositive rates in patients with other diseases were 22.5% for CF, 52.5% for LIT, 12.5% for IHA and 67.5% for ELISA. The results suggest that the test of choice for giardiasis was CF with 88% specificity, nevertheless this test showed low sensitivity (36%). Other two tests, ELISA and LIT were more sensitive than CF with percent sensitivity of 72 and 58 respectively, but these two tests had severe disadvantages in being less specific with percentage specificity of 48 and 60 respectively.

Leucocyte migration agarose test to study cell-mediated immunity in amoebiasis.

The leucocyte migration agarose test (LMAT) was used to measure quantitatively the magnitude of cell-mediated immunity (CMI) in 35 patients with amoebic liver abscess and 22 healthy controls. LMAT was positive in 65.7% and 85.7% of patients with amoebiasis in the presence of 200 microgram and 400 microgram of the amoeba extract respectively, whereas the test in all 22 healthy controls was negative. Time course studies showed that within 10 days after the onset of clinical illness, only 1 of 4 patients was positive. Thereafter the percentage positivity was raised, especially when 400 microgram antigen was used. Maximum CMI response was apparent between 31-60 days after the onset of clinical illness. The indirect haemagglutination (IHA) test showed that all patients and 5 of 22 healthy controls were positive. These was no correlation between IHA titers and the magnitude of LMAT reaction.

Cellulose acetate membrane precipitin (CAP)test in amoebiasis using antigens from four different strains of Entamoeba histolytica.

A simple cellulose acetate membrane precipitin (CAP) test was evaluated against immunoelectrophoresis (IEP) test using saline extract from 4 different strains (HK-9, HT-10, HT-12 and HT-31) of axenically grown Entamoeba histolytica as the antigens. All 81 sera from patients with amoebic liver abscess were positive in the IEP against the antigens from all 4 strains. With the CAP test the number positive against antigens from HK-9, HT-10, HT-12 and HT-31 were 79,77,79 and 71 respectively Sera from 100 blood donors were negative by both IEP and CAP tests against antigens from all 4 strains. Comparison between the number of precipitating bands demonstrated by either IEP or CAP test showed that strain HT-12 was the best source of antigen in exhibiting significantly more number of precipitating bands, strain HT-31 was the poorest. The strain HT-10 was comparatively superior to strain HT-31 in the CAP test whereas in the IEP test strain HK-9 and HT-31 were both inferior to strain HT-10.

Humoral immune response of cats to Paragonimus infection.

The time course of humoral immune response to Paragonimus siamensis was studied in 10 cats experimentally infected with either 30 or 60 matacercariae and the antibody produced was detected by enzyme linked immunosorbent assay (ELISA), complement fixation test (CFT), and immunoelectrophoresis (IEP) test. With ELISA and CFT, antibodies was detected as early as 2nd week after infection and the cats remained positive throughout the 12 week period of observation. In contrast, the IEP test was persistently negative. With respect to sensitivity, both ELISA and CFT are equally sensitive but the mean ELISA titer was consistently higher than that of CFT. THe magnitude of the antibody response appeared to be related to duration of the infection but not to the infective dose and the number of worms recovered. There was variability in titers among cats infected with equal numbers of metacercariae. The tests can not be used for differential diagnosis of infections by P. siamensis and P. heterotremus because of the cross-reaction. Such cross-reaction did not not occur against unrelated parasites including hookworm. Toxocara cati and Spirometra mansoni.