Immunogenetic factors involved in the pathogenesis of distinct clinical manifestations of schistosomiasis japonica in the Philippine population.

Immunogenetic factors were studied in 60 patients with schistosomiasis japonica in the Philippines, of whom 15 were characterized by marked hepatosplenic lesions and 45 characterized by cerebral symptoms. Immune responsiveness of the patients to schistosomal antigen was measured by T cell proliferation in vitro, and their HLA-A and -B specificities were typed. All but one hepatosplenic patients showed strong immune responsiveness to the schistosomal antigen, whereas both low and high responders were observed in the cerebral patients. A significant association between HLA-B40 and high responders to the schistosomal antigen was observed (P = 0.0458), and this HLA specificity was increased in frequency in the hepatosplenic patients. HLA-B16 was not observed in the hepatosplenic patients, but was common in the cerebral patients (26.5%) (P = 0.0255), and this HLA specificity was commoner in the low responders than in the high responders. These observations suggest that an HLA-linked gene governs the clinical manifestations of human schistosomiasis japonica by controlling immune responsiveness of the infected hosts to the schistosomal antigen.

An attempt to study the case fatality rate in Schistosoma japonicum infection in the Philippines.

As a sequence to a cross-sectional study on the clinical gradient of the disease by Pesigan et al., (1965) a follow-up of the subjects in that study was made after 12 years. Of the 135 untreated cases followed up, 23 (17.04%) died from various causes of which 12 (8.89%) had signs and symptoms attributable to schistosomiasis as the immediate cause or one of the main causes of death. This occurred in 1 to 11 years with an average of 5 years, which corresponds roughly to 1.78% of the infected cases per year. This is considered a conservative estimate because in the other deaths due to other diseases, schistosomiasis is a contributory cause. A diminishing severity of the disease was observed among the surviving patients which could imply that they must have developed some degree of immunity to the disease.

Longitudinal study on Schistosoma japonicum infections in the Philippines. 4. Effect of mass-chemotherapy with praziquantel on incidence at Dagami, Leyte.

The effect of selective mass-chemotherapy with praziquantel at a dose of 60 mg/kg a day on the annual incidence of Schistosoma japonicum infection was examined by a follow-up study of 1,800 school children enrolled at 9 primary schools at Dagami Area, Leyte, Philippines. The incidence rates before mass-chemotherapy were 22.2, 24.2, 26.9, 9.6 and 28.4% in a period from school year (SY) 1975/76 to 1979/80, respectively, where the incidence of 9.6% in SY 1978/79 was probably due to extraordinary drought in the previous year. Egg positives including village people were treated and the numbers treated yearly were 22, 943, 273, 300 and 341 from SY 1978/79 to SY 1982/83, respectively. The incidence was reduced drastically as early as one year following the start of mass-chemotherapy, i.e., 8.4, no survey, 6.8 and 15.4%, from SY 1980/81 to SY 1983/84, respectively, and which demonstrated the efficacy of mass-chemotherapy on reduction of transmission.

Evaluation of ELISA with ABTS, 2-2'-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate of peroxidase and its application to the diagnosis of schistosomiasis.

A micro-technique of enzyme-linked immunosorbent assay (ELISA) using ABTS, 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid), as a substrate for horseradish peroxidase (HRP) conjugate was studied. In a comparative study among 4 substrates, namely; 5-aminosalicylic acid (5AS), O-phenylenediamine (OPD), O-tolidine (OT) and ABTS, for HRP in terms of sensitivity, it was found that ABTS was the most sensitive, stable and the best in visuality by its bluish-green color. The precise reproducibility of ELISA readings was attained by making correction of observed absorbance with positive reference serum. Sera from 50 patients positive for S. japonicum (Sj) egg and from 50 healthy residents in non-infested areas were diluted at 1:200 and examined by this procedure of ELISA against crude egg antigen. The upper limit of 99% critical range of the absorbance among negative sera was 0.080. The reactions of egg positive cases were beyond this value, being 0.505 at the minimal absorbance (1.259 +/- 0.278), and were easily distinguished from negatives even by naked eyes. Cross-reactivity to Sj egg antigen was studied using sera from 75 cases with 22 kinds of varied parasitic infections. Among them, S. mansoni and S. haematobium infections showed higher absorbance while the others did a little higher than the normal range. The highest absorbance shown by a Trichobilharzia brevis case was 0.177. The results indicated that micro-ELISA technique with ABTS developed in the present study is applicable to the field survey because of its high sensitivity, specificity and reproducibility.

Phase III clinical trials with praziquantel in S. japonicum infections in the Philippines.

Following successful Phase II clinical studies with praziquantel (2-cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino [2,1-a]isoquinolin-4-one, EMBAY 8440, Biltricide), expanded field trials of Phase III were conducted in Mindoro Oriental, Leyte and Davao Norte, to reassess efficacy, safety and acceptability of the drug on a larger scale. A total of 6,134 cases were treated with the best tolerated and effective dose of 60 mg/kg bwt which for practical purposes was given in 2 instead of 3 divided doses as applied in the earlier trials. Two aliquots of one stool from each individual were examined quantitatively by a modification of the thick smear method one week before, six and twelve months after treatment. Although 67.8% were noted to have drug-related side effects, most were mild to moderate, with only 1.2% considered as severe. Severe reactions consisted mainly of colicky abdominal pain, occurring about 1 h after drug intake, usually accompanied by fever, sweating, urge to defecate, and occasionally by discharge of bloody stool. These reactions, however, required only symptomatic treatment with antispasmodics and antipyretics. Stool follow-up 6 months after treatment showed 89.2% of EPG (eggs per gram of faeces) negativity, with an overall egg reduction of 91.1%. Twelve months after treatment, practically the same results were obtained with 87.5% still negative cases and an egg reduction of 90.5%. This study confirms safety and efficacy of the drug as well as its acceptability when given on a community wide scale.

Evaluation of micro-ELISA for schistosomiasis japonica using crude egg antigen.

Procedures of micro-ELISA for detecting antibody of Schistosoma japonicum infection were improved by using crude egg antigen, peroxidase-labeled antibody and O-phenylenediamine on a micro-ELISA plate (M129A, Dynatech). Reactions were performed with 0.1 ml of reagents in 0.3 ml wells at each step and 0.3 ml of substrate was placed at the final procedure. The endpoint of reaction was defined as the upper limit of 99% critical range of absorbance in negative sera at 1:40 dilution which was approximately twice the absorbance of a pooled negative serum at 1:40. Using this endpoint, appropriate concentrations of antigen and conjugate were determined. Cross-reactions of egg antigen were observed with sera at 1:40 from the infections with other schistosomes, Trichobilharzia brevis, Fasciola hepatica, Echinococcus multilocularis and Trichinella spiralis were diminished at 1:200 serum dilutions except for other schistosomes. Among 177 egg positives, 171 (96.6%) showed the titer of 200 or higher while 67 old cases at Kofu, Japan showed low titers where 22 (31.9%) were lower than 40, 44 (63.7%) were between 40 and 160 and 3 (4.4%) were 320 or higher. The proven non-infected controls of 93 cases from Leyte and Manila, Philippines, Tokyo and Kofu, Japan were all negative. The result of ELISA for schistosomiasis japonica by crude egg antigen was satisfactory after standardization and stabilization of the procedures which were considered to be as important as using defined antigens.

Longitudinal study on Schistosoma japonicum infections in the Philippines. 3. Incidence and environmental modification at Dagami, Leyte.

The yearly change of incidence of schistosomiasis japonica was observed among 1800 children enrolled in 9 schools situated within a 5 km radius around Dagami Poblacion, Leyte, Philippines from School Year (SY) 1974/75 to 1979/80. The purpose of this observation was to know the effect of environmental modification undertaken in about 50 ha of snail-infested and abandoned rice field which is the most depressed and central portion of this project area. Children were examined by egg detection from stool and by circumoval precipitin test. Coverage of examination among the school population ranged from 40 to 80% yearly and the ratio of re-examination after about 1 year was from 30 to 60%. The annual incidence in the different schools differed greatly from 12.15 to 42.86% in contrast to the annual prevalence which differed only slightly. There was corresponding increase of incidence with age from 18.6% at age 7 to 33.9% at age 12 while no significant difference was observed between sexes. The overall annual incidence rates for 9 schools during the 5-year follow-up period (SY 1975/76 to 1979/80) were 22.2, 24.2, 26.9, 9.6 and 28.4%, respectively. Those of Dagami Central II, the nearest school to the reclaimed site, were 15.7, 18.2, 19.3, 5.2 and 11.6%, respectively, for the same period. Obviously, the incidence was increasing while the reclamation was going on except in SY 1978/79. Analysis of the sudden drop of incidence in that year in all the 9 schools showed that it was not due to the effect of reclamation, but was most likely due to scanty rainfall in the previous year (1977). These observations indicated that small scale environmental modification had doubtful effect on outlying endemic areas in reducing the danger of Schistosoma infection but may have a little effect on the immediate surroundings as shown by a lesser elevation of incidence after the abrupt decrease at the nearest school as compared to those of schools located farther from the reclamation area.

Annual fluctuation of Schistosoma japonicum infection in field rats, Rattus rattus mindanensis, in Dagami, Leyte, Philippines.

The annual fluctuation of Schistosoma japonicum infection in field rats, Rattus rattus mindanensis, in Leyte, the Philippines was investigated. One hundrend live-traps were set for one week every month from February, 1978 to February, 1979, in and around Central Elementary School II, Diagami, Leyte. Out of 199 rats, 163 (81.9%) were infected. The prevalence rate of S. japonicum infecton in rats was high during the relatively dry season, April to July, 1978. Meanwhile, the prevalence rate of schistosome cercariae in Oncomelania quadrasi of the study area increased during the relatively rainy season, October, 1978 to February, 1979. It is supposed that miracidia hatched from eggs discharged in the feces of rats during the relatively dry season would encounter snails, following which the emerging cercariae would penetrate the mammalian hosts, such as humans and rats, during the relatively rainy season. This might be the transmission dynamics of schistosomiasis in the endemic areas of Leyte. It is also suggested that this annual prevalence rate could be used as a parameter to formulate and evaluate control works. No infection was established in the youngest age group, the weanling. Then, the prevalence increased gradually by age. All rats of the older age group were infected. According to age composition, the field rat had no definite breeding season in Leyte. COP reaction was weak in sera of infected rats, but strongly exhibited in sera from rats possessing eggs in the spleen. Consequently, field rats not only maintain the infection as reservoir hosts, but also are an important source of infection as definitive hosts in the endemic areas of Leyte Island. Specifically, the rat role must be stressed, should human schistosomiasis ever be controlled.

Detection of antibodies in Schistosoma japonicum infections by a micro-technique of enzyme-linked immunosorbent assay (ELISA).

Antibodies in Schistosoma japonicum infections were successfully detected by micro-technique (0.3ml/well) of ELISA on polystyrene microtiter plates using peroxidase labelled anti-human IgG and 5-amino salicylic acid as conjugate and substrate, respectively. Serum samples collected from 22 proven patients in Leyte, Philippines showed titers of higher than 1:960 in 18 cases, 1:240 in 2 cases and 1:120 in 2 cases whereas titers of 22 proven negative sera collected in Tokyo were less than 1:15. Though the reaction was read by spectrophotometry in the present study, the difference of reactions between positive and negative sera was so clear as to be recognizable readily by visual readings.

Longitudinal study on Schistosoma japonicum infections in the Philippines. Incidence and prevalence among school children in Dagami Area, Leyte.

The annual incidence of Schistosoma japonicum infection was calculated from the record of the yearly examination among school children of Dagami Area, Leyte, Philippines. Data were processed mainly by the computer. Children were examined for eggs by the merthiolate formalin concentration technique and by the circumoval precipitin test (COPT) once a year in 3 consecutive school years (SY), SY 1974/75, 1975/76 and 1976/77. About 600 to 700 children were examined yearly and the ratios of cases rechecked after 1 year were about 30%. A method to calculate the overall incidence in children who were examined at different intervals, was newly established. The incidence appeared to rise rapidly in the survey period, being 25.59% and 41.30% by fecal examination and 10.74% and 21.27% by COPT in SY 1975/76 and 1976/77, respectively. From the combined results of fecal examination and COPT, the reliable values of incidence, however, were shown to be stable during 2 years such as 22.06% and 24.21%, respectively. The incidence estimated by the age prevalence data was 20.25% in SY 1976/77 by the combined results and was found to be lower than the directly calculated value.

Preliminary clinical trials with praziquantel in Schistosoma japonicum infections in the Philippines.

Praziquantel, a new antischistosomal compound, was tested for tolerance and efficacy against placebo in two double-blind clinical trials in Philippine patients infected with Schistosoma japonicum.The compound was given orally at a dose of 3 x 20 mg/kg at intervals of 4 hours to a total of 82 patients-some without advanced disease and some with hepatosplenic involvement. A total of 43 patients received placebo. In a single-blind trial, 42 patients were given a single oral dose of 50 mg/kg. Monitoring of vital organ functions included comprehensive laboratory tests and serial electrocardiograms. In 38 patients with hepatosplenic involvement due to advanced stages of infection, serial electroencephalograms were additionally recorded. No toxic effects were observed in any of these examinations.Undesirable side effects occurred in 53% of the patients given 3 x 20 mg/kg and in 70% after a dose of 1 x 50 mg/kg. They consisted mainly of abdominal discomfort, fever, sweating, and occasionally giddiness, but in general were transient and mild. At 6 months post-treatment, 60 of 75 patients treated with 3 x 20 mg/kg and 29 of 41 treated with 1 x 50 mg/kg were completely negative for eggs. At 12 months post-treatment, 25 of 33 and 14 of 26 patients in the two treatment groups were cured. Thus the divided dosage gave a superior therapeutic result. Praziquantel proved to be free of major toxicity, and was well tolerated, highly effective, and easy to administer. Confirmation of results in extended trials may soon permit large-scale treatment.

Final localization of Schistosoma japonicum in the lungs of field rats, Rattus mindanensis, in Leyte, Philippines.

Among 45 specimens of Rattus mindanensis (Hearns, 1,905) caught in an endemic area of schistosomiasis japonica in Leyte, Philippines, 39 (86%) were found to be positive for Schistosoma japonicum. Lung examination of 24 autopsied rats revealed a high incidence of pulmonary infection and the flukes were found in 20 rats (83%). In 24 (73%) out of 33 rats, the flukes were seen in the hepatic portal vein. Only 29% of the rats were positive for Schistosoma ova in the intestinal wall. The average number of living worms was much greater in the lungs (19.2) than in the liver (12.4). However, fewer fully developed worms of oviposition stage were found in the lungs than in the liver. In the lungs widespread and massive nodules containing living worms and distinct lesions due to embolism were observed histopathologically.