Serologic and ultrasonographic parameters of praziquantel treatment of hepatic fibrosis in Schistosoma japonicum infection.

We describe the parameters useful in evaluating the development of hepatic fibrosis in Schistosoma japonicum infection, as well as its improvement after treatment with praziquantel (PZQ). Various serologic parameters and ultrasonographic images were examined, and their changes were monitored using rabbits infected with 200 or 300 cercariae of S. japonicum. Infected rabbits were administered one oral treatment of PZQ at a dosage of 100 mg/kg at 6, 12, or 24 weeks after infection. Histopathologic examinations revealed that PZQ had a strong and rapid effect, even on damage that developed long after the infection. The improvement of moderate hepatic fibrosis that developed over 24 weeks after infection was also detected by histopathologic examinations. The serum level of total bile acid was the most sensitive parameter in evaluating the severity of hepatic fibrosis and its improvement after treatment with PZQ. The level of serum procollagen-III-peptide was also useful in evaluating the development of hepatic fibrosis, but not in its improvement. Ultrasonography revealed specific echogenic bands and nodules according to the progress of granuloma formation and fibrosis, and the reversal of these changes could also be observed after treatment with PZQ.

A comparison of the antischistosomal effect of levo- and dextro-praziquantel on Schistosoma japonicum and S. mansoni in mice.

Praziquantel (PZQ) is a racemic compound composed of equal proportions of its optical isomers, levo- and dextro-PZQ. The efficacy of these compounds was compared with that of PZQ in mice infected with Schistosoma japonicum or S. mansoni. Mice were given 50, 2 x 50 (on consecutive days), 500, or 2 x 250 mg/kg of each compound orally 5 weeks after infection. Significant reduction of worm recovery was observed in S. japonicum infection 30 days after treatment with 2 x 50, 500, or 2 x 250 mg/kg of levo-PZQ, whereas no therapeutic effect was demonstrated with dextro-PZQ. Percent reduction in worm burden in mice treated with levo-PZQ was significantly higher than in those with PZQ at a dosage of 2 x 50 mg/kg (67.9% vs. 34.5%). Neither eggs in feces nor miracidial hatching of eggs from the livers and intestines were observed in mice treated with levo-PZQ. In S. mansoni infection, levo-PZQ showed no significant schistosomicidal effect compared with PZQ and dextro-PZQ, although there was reduction in egg counts.

Schistosoma japonicum and S. mansoni: ultrastructural damage in the tegument and reproductive organs after treatment with levo- and dextro-praziquantel.

Ultrastructural observations were made of changes in the tegument and reproductive organs of Schistosoma japonicum and S. mansoni from ICR mice after treatment with praziquantel (PZQ), levo-PZQ, and dextro-PZQ at a single oral dose of 500 mg/kg body weight. No marked difference in types and extent of lesions of the tegument of S. japonicum was found between the compounds regardless of the time of worm recovery after treatment. This was equally true of S. mansoni. Degeneration of the testis, ovary, and vitelline gland of S. japonicum was more prominent in worms administered PZQ and levo-PZQ than in those receiving dextro-PZQ. In S. mansoni, extensive regression of the reproductive organs was observed in male and female worms treated with PZQ and dextro-PZQ, while no serious damage was seen in worms treated with levo-PZQ.

Ultrasonographic and serologic abnormalities in Schistosoma japonicum infection in Leyte, the Philippines.

We have identified specific ultrasonographic changes in Schistosoma japonicum-infected patients associated with serologic indicators of general liver function. An ultrasonographic examination concomitant with hematologic and biochemical serum analyses was performed on 102 patients at the Schistosomiasis Hospital in Leyte, The Philippines. The ultrasonographic liver images were classified into four patterns, according to the development of periportal fibrosis and the patterns of echogenic bands. Eleven cases with a long-term infection showed typical septal formation (network pattern). Other ultrasonographic changes in the portal system, such as the severity of splenomegaly, did not correlate with the age of the study patients or the duration of their infection; however, the production of collateral vessels was clear in the group of older patients. Among various hematologic and biochemical serum indicators of liver damage, the serum levels of total bile acid (TBA) and procollagen-III-peptide (P-III-P) strongly correlated with the development of hepatic fibrosis and protal hypertension. These findings suggest that the ultrasonographic liver patterns classified here, along with the changes in serum levels of TBA and P-III-P, provide useful indicators for field monitoring of S. japonicum infection.

Improvement of ultrasonographic and serologic changes in Schistosoma japonicum-infected patients after treatment with praziquantel.

We previously reported ultrasonographic and serologic abnormalities in 102 patients infected with Schistosoma japonicum in Leyte, The Philippines. These patients were subsequently treated with praziquantel (3 x 20 mg/kg), and changes in ultrasonographic images and the serum levels of liver function markers in 52 patients were followed up every three months for a period of 17 months. Improvement in the thickening of the portal vein wall and the intensity of echogenic bands was detected six months after treatment with praziquantel. The level of splenomegaly was also reduced in 42 patients who originally did not show the production of collateral vessels. A significant decrease in the serum total bile acid (TBA) level was detected in all patients six months after treatment with praziquantel. However, significant ultrasonographic changes could not be detected in the patients classified as type 3, with severe hepatic fibrosis caused by the long-term infection. These results clearly show that ultrasonographic examination, along with data on the serum TBA level, provides a sensitive tool to monitor the severity of hepatic fibrosis and portal hypertension caused by S. japonicum infection, as well as the improvement resulting from praziquantel treatment.

Oral itraconazole and topical miconazole with débridement for Acanthamoeba keratitis.

We treated three patients who had Acanthamoeba keratitis with oral itraconazole, a new antifungal agent, topical miconazole, and surgical débridement of the lesion. In these patients, healing and regression of the keratitis began six or seven days after initiation of oral itraconazole and miconazole 0.1% eyedrops (every hour during the day). The clinical signs of corneal infection disappeared after nine weeks in Patient 1, after five weeks in Patient 2, and after eight weeks in Patient 3. Visual acuities improved markedly from hand motions to 20/30 in Patient 1, from counting fingers to 20/16 in Patient 2, and from hand motions to 20/40 in Patient 3. In these patients, no systemic or topical signs of toxicity or adverse reactions were noted during the course of treatment.

Degenerative changes in the reproductive organs of female schistosomes during maintenance in vitro.

Degenerative alteration of the reproductive organs of female schistosomes in correlation with the change in egg-laying rate of schistosome pairs in vitro was studied by electron microscopy. The production of normal eggs by adult S. japonicum pairs decreased after 4 days in vitro followed by an increase of abnormal egg laying up to day 8. In S. mansoni, the yield of both normal and abnormal eggs decreased gradually from the start of maintenance in vitro in spite of a much higher pairing rate than in S. japonicum. The vitelline gland of 14-day in vitro-maintained S. japonicum stained with Fast red B, while that of S. mansoni did not. The ovary of both species exhibited regressive features after 14 days of maintenance in vitro. Ultrastructural examination showed that the vitelline cells and oocytes of S. japonicum and S. mansoni had already lost their structural integrity after 2 days in vitro and continued to exhibit signs of structural degeneration throughout the 14-day in vitro maintenance period. The regressive changes in reproductive potential of female S. mansoni maintained in vitro for 4 days could be reversed by surgically implanting the parasites into mouse mesenteric veins.

Schistosoma japonicum: ultrastructural changes in the tegument during cercaria-schistosomulum transformation.

The tegumental changes of Schistosoma japonicum cercaria were initiated when introduced into NCTC 109 containing antibiotics for axenizing. In some regions the tegument was covered with a penta- or heptalaminate membrane, and the surface coat changed into an electron-dense, amorphous material. Large vacuoles limited by a multilaminate membrane were evident in the tegumental cytoplasm, and some appeared to join or open to the tegument. After 4 h incubation in NCTC 109 containing 50% fresh rabbit serum, the outer tegumental membrane of schistosomulum became heptalaminate. The amorphous material was still present on the surface of the tegument. Within 72 h the tegument was covered with a heptalaminate membrane over a greater part of the body, and the amorphous material was very reduced. Large vacuoles in the tegument disappeared and tegumental folds developed in some regions. No amorphous material existed on the surface of 10-day schistosomulum and the outer membrane of the tegument was heptalaminate in almost all regions of the body.

Morphological alterations of Schistosoma japonicum associated with the administration of amoscanate.

Morphological alterations of Schistosoma japonicum induced by the administration of a curative dose (20 mg/kg) of the schistosomicide, amoscanate (4-isothiocyanate-4'-nitrodiphenylamine, CGP4540), were studied. Worms from amoscanate-treated ddY mice exhibited remarkable changes on the surface of male and female worms, as well as in the vitelline cells of females. Various types of lesions, such as swelling and ballooning of the tegument, constriction of folds and channels, disruption of sensory receptors and exfoliation of surface layers, were prominent in many areas of the worm body. These alterations were variable in different worms recovered from the same host. The extent of the lesions was not dependent on the period of recovery after treatment.

Schistosoma japonicum: the effect of serum fractions precipitated by ammonium sulfate on viability of schistosomula.

The effect of serum fractions precipitated by a saturated ammonium sulfate solution on viability of Schistosoma japonicum schistosomula was investigated. Axenized cercariae were incubated for 10 days at 37 degrees C in NCTC 109 supplemented with the 34%, 40%, 50%, 62% and 68% fractions of human or rabbit serum in an equivalent concentration to 10% serum. Only larvae cultured in media with the 68% fraction of human or rabbit serum retained the transparent feature and active motility, while the larvae cultured in other media became opaque and immotile. This was also true in media with human serum albumin or fatty acid-free human serum albumin at concentrations of 1.0 and 10.0 mg/ml of NCTC 109. On 10-day culture a part of larvae in the medium with the 68% fraction of human serum began to feed on red blood cells and the intestine was nearly closed by 21 days. These results suggest that serum fraction of 68% ammonium sulfate consisting of albumin is an important constituent in supporting the further growth of schistosomula in vitro.

Ultrastructural observations on damage to schistosomula of Schistosoma japonicum by mouse neutrophils in vitro.

Ultrastructure of schistosomula of Schistosoma japonicum was studied in relation to the effect of mouse neutrophils in vitro. In the presence of antibody and complement, damage to the schistosomular tegument covered with many neutrophils began to appear within 1 h incubation. Exfoliation of the granular cytoplasm of the tegument, disorganization of the muscle layers and vacuolation in the inner tissues were seen in a part of neutrophil-attached parasites by 2 h incubation. However, the majority of schistosomula with fewer cells retained their integrity till 16 h. The cytochemical examination of schistosomula incubated with mouse neutrophils for 1 and 2 h demonstrated clearly the localization of the peroxidase activity on the surfaces of the cells and the parasites.

In vitro cultivation of Metastrongylus apri.

The 3rd stage larvae of Metastrongylus apri were successfully cultured in vitro to the nearly maturity adult worms which possessed the sperm and immature eggs, though the worms were smaller in size than those grown in vivo. The most excellent medium for the development was relatively simple and consisted of chemically defined NCTC 109 supplemented with 20% calf serum, penicillin (200 unit/ml) and streptomycin (100 micrograms/ml). Standing culture with a larval concentration of 100 to 200 worms per tube containing 2 ml medium and wih a gas phase of 5% CO2 in air at 37 to 28 degrees c was found to be sufficient twice a week using aseptic techniques. Under these conditions a relatively uniform development was obtained, attaining 85 +/- 6.7% of larvae to the 5th stage.

Conversion of schistosome cercariae to schistosomula in serum-supplemented media, and subsequent culture in vitro.

Both Schistosoma japonicum and S. mansoni cercariae, axenized by washing in NCTC 109, transformed to schistosomula when incubated directed in NCTC 109 containing 2--50% human or 50% rabbit serum at 37 degrees C. The resultant schistosomula were grown in vitro to adults which mated, although the females did not produce eggs. The cercaria-schistosomulum transformation took place at a slower rate in heat inactivated serum than in unheated serum.