Distribution of [3H]cholesterol-labelled liposomes with or without praziquantel in mice infected with Mesocestoides corti (Cestoda) tetrathyridia.

The anthelmintic drug praziquantel (PZQ) has a short half-life in the circulation, necessitating repeated daily administration of PZQ for the therapy of larval stages of cestodes. The effect of incorporation of PZQ into multilamellar liposomes on their biodistribution in Mesocestoides corti (syn. M. vogae) infected mice has been examined using [3H]cholesterol as a liposomal marker. Incorporation of PZQ significantly increased the average size of liposomes with 70.3% of [3H]lip.PZQ particles up to 1.9 microm, whereas higher portion of [3H]liposomes (66.3% of total) were of smaller (up to 1.3 microm). Both liposome preparations were given intraperitoneally to avoid rapid sequestration in the liver. There were significant differences between [3H]liposomes and [3H]lip.PZQ-associated radioactivity in peritoneal adherent cells, liver- and peritoneal larvae, liver, spleen and lymph nodes within 16 days of examination. The highest uptake (about 2-fold more [3H]lip.PZQ than [3H]liposomes from the total dose) was found in peritoneal cells on day 1 post therapy (p.t.) followed by a rapid decline. The kinetic of decline in these cells recovered on day 1 p.t. was studied also in vitro. Disappearance of the marker due to the breakdown of liposomes and efflux of lipids and PZQ from cells was slower for [3H]lip.PZQ in comparison with drug-free liposomes and was not completed after 4 days-incubation. Significantly increased levels of radioactivity, more in [3H]liposomes treated groups, were recorded in the liver- and peritoneal larvae between days 8-16 p.t. indicating re-utilization of cholesterol by the larvae. The data suggest that incorporation of PZQ into liposomes contributes to the enlargement of liposome average size and slows down their degradation in phagocytosing cells. In this respect, these cells could serve as the secondary circulating depots for PZQ releasing it slowly to the circulation.

Human schistosomiasis in Puerto Rico: reduced prevalence rate and absence of Biomphalaria glabrata.

A combined epidemiologic and malacologic survey of schistosomiasis in Puerto Rico was carried out in areas where previous surveys had reported the prevalence of the disease. This limited survey, with 495 persons examined, found a low prevalence (0.6%) of Schistosoma mansoni infections. The infections were restricted to three people more than 36 years of age. No infections were detected in children 16 years of age or less, and this cohort comprised 57.8% of the study group. Malacologic surveys of the four streams, 10 rivers, and eight lakes throughout the island revealed the absence of intermediate host Biomphalaria glabrata and the presence of Thiara granifera, a competitive species of B. glabrata and the predatory snail Marisa cornuarietis. We believe that the absence of B. glabrata is the primary reason for the sustained reduction in the prevalence of schistosomiasis in Puerto Rico.

Impact of antifibrotic treatment of the course of Schistosoma mansoni infection in murine model.

Administration of an antifibrotic agent as an adjunct to antihelmintic treatment with the objective of morbidity reduction was investigated in the murine schistosomiasis mansoni model. Antifibrotic, beta-aminopropionitrile treatment has a profound effect on the cellular matrix composition of the liver granuloma of Schistosoma mansoni infected mice when given alone, resulting in increase macrophage infiltration. These macrophages, in response to stimulation with soluble egg antigen or lipopolysaccharide produced elevated levels of nitric oxide but low levels of tumor necrosis factor alpha compared to untreated infected mice. This also correlated with reduced liver granuloma size. In spite of low numbers of eggs in the liver, mice receiving a combine treatment had a high level of resistance to a challenge infection compared with mice receiving only praziquantel. Those mice also exhibited a reduced lymphocyte proliferative response, similar to that of infected untreated mice. Antifibrotic treatment has an impact on the dynamic of the cellular nature of granulomas and impacts on the host immunity of infection.

Immune response of mice infected with Schistosoma mansoni is modulated by antifibrotic treatment.

Mice infected with Schistosoma mansoni treated with beta-aminopropionitrile (BAPN), an antifibrotic agent, and the antischistosomal drug praziquantel (PZQ) were resistant to challenge for up to 5 weeks post-treatment. The combined treatment resulted in profound changes to the liver granuloma cell matrix and the composition and function of the cellular infiltrate. Although granulocytes always predominated in the infiltrate, the proportion of the cells which were macrophages was higher in mice treated with BAPN alone (39.2%) than in infected mice which were untreated (15.2%) or treated with PZQ alone (12.4%), and much higher than in mice given the combined treatment (1.8%). Two products associated with macrophage activation and cytotoxicity [tumour necrosis factor-alpha (TNF-alpha) and nitric oxide] were only detected in mice harbouring a patent infection and there was a strong positive correlation between the concentrations of each. The relatively low TNF-alpha concentrations in BAPN-treated mice seemed to be associated with the relatively small granulomas observed in these mice. BAPN treatment also led to changes in the proliferative response of the treated mice's macrophages to mitogen and soluble schistosome-egg antigen and in spleen cellularity; these changes are probably associated with the resistance to challenge infection observed in mice given BAPN with PZQ. It is clear that BAPN treatment changes the dynamics of the delayed-type hypersensitivity response within the granuloma and that this impacts on other immunological sites. How this relates to the maintenance of post-treatment resistance to a challenge infection has still to be elucidated.

Reduction in tissue egg load and maintenance of resistance to challenge in mice infected with Schistosoma mansoni, following combined treatment with praziquantel and an antifibrotic agent.

Antifibrotic therapy with beta-aminoproprionitrile (BAPN), when combined with praziquantel (PZQ) treatment of mice infected with Schistosoma mansoni, led to a marked change in the temporal pattern of parasite egg excretion. The rate of egg excretion was higher and terminated sooner and the tissue egg load was smaller in mice treated with BAPN and PZQ than in mice treated with PZQ alone. Some of the mice that received the combined treatment were challenged 2 or 5 weeks after PZQ treatment; they retained a high level of resistance to challenge, similar to that observed in untreated, infected mice. In contrast, mice given only PZQ were as susceptible to challenge as the uninfected control mice. The activity of peritoneal macrophages, as measured by their production of hydrogen peroxide, nitric oxide and tumour necrosis factor alpha in vitro, was only elevated in cells obtained from untreated, infected mice. These results demonstrate that modulation of granuloma formation by combined antifibrotic/PZQ therapy significantly changes the dynamics of egg granuloma formation and attenuates the host's resistance to challenge infection.

Schistosoma mansoni eggs as a target for praziquantel: efficacy of oral application in mice.

A study was undertaken to investigate the effect of administering a double oral dose of praziquantel (PZQ), given 9 days apart, on the egg viability in Schistosoma mansoni infected mice. The viability of eggs trapped in the tissues and those excreted in the faeces was determined using morphological (oogram) and function (egg hatching) criteria. Nine days after the first dose of PZQ, 90.5% of eggs excreted in the faeces were judged to be viable by the egg hatch method, as were 98.0% of eggs excreted from the control mice. However, only 44.0% of the eggs isolated from the intestine of PZQ treated mice were viable compared with 82.5% in the pretreatment group or 93.0% in the control group. Similar results were obtained for viability of eggs isolated from the intestine, by both the hatching and oogram methods. There was a dramatic reduction in the viability of eggs in liver tissue 9 days after the first dose of PZQ (13.0%) when compared with either the untreated controls (85.3%) or eggs in the livers of mice prior to PZQ treatment (80.0%). Following a second dose of PZQ (69 DPI), no eggs were detected in the faeces by 78 DPI, and eggs in the tissues were no longer viable. These data confirm the susceptibility of mature eggs of S. mansoni to a single dose of PZQ, but also demonstrate that the remaining immature eggs are undamaged and mature in the tissues of infected mice. Administration of a second dose of PZQ, 9 days after the first is suggested as an alternative treatment strategy assuring killing of all the schistosome eggs.

Alkaline phosphatase as marker of Schistosoma mansoni egg viability.

A reliable assessment of the viability of schistosome eggs trapped in host tissues is difficult. The use of a coupling azo dye method for the detection of alkaline phosphatase (AlP) in Schistosoma mansoni ova was found to be a specific and sensitive method for differentiating between viable and dead eggs, and can be used in both immature and mature eggs. In fully developed miracidia within an egg, AlP activity was demonstrated in germ cells and in the sensory endings neural cells. The embryonating miracidia displayed AlP activity on the body surface and in von Lichtenberg's envelope. The alkaline phosphatase test for egg viability shows increased sensitivity when compared with the more conventional Oogram and Hatching tests.

Schistosoma mansoni: determination of the sex of cercariae using a nonradioactively labelled DNA probe.

Cercarial sex determination protocol which utilizes nonradioactive nucleic acid labelling with digoxigenin (DIG)-11-dUTR, offers a rapid "clean" identification of the sex of Schistosoma mansoni cercaria. DIG labelled pW1-DNA (plasmid female-specific genomic DNA clone--W1) hybridized with the native cercarial DNA, and hybridization signals are visualized with Lumigen PPD. Results obtained are comparable with those obtained with the 32P labelled pW1.

[The clinical picture of imported malaria and its relation to P. falciparum parasitemia]. / Klinický obraz importované malárie a jeho vztah k parazitémii P. falciparum.

The paper summarizes the results of clinical examinations of imported malaria in 48 patients, semi-immune foreigners infected with P. falciparum and P. vivax/P. ovale. Of the objective manifestations, hepatosplenomegaly was predominant in P. falciparum infections (34%), whereas in P. vivax infections its occurrence rate was 19%. Body temperature over 38 degrees C with shivering had a higher frequency in P. vivax (81%) compared to P. falciparum (41%) infections. Pathologically altered hematologic values, particularly mild to medium increase in sedimentation rate, were found in all patients with P. vivax/P. ovale, while in patients infected with P. falciparum only in those whose parasitemia exceeded 10,000 trophozoites in 1 microliter blood. Reduced values of hemoglobin, hematocrit, and mean erythrocyte volume are indicative of microcytic anemia of mild degree. Biochemical examinations yielded slightly increased values of the thymol turbidity test and mild reduction of serum proteins at simultaneous increase of albumins (8% of the patients examined). Of the parameters of humoral immunity, there was a rise in immunoglobulin values concerning particularly IgG and IgM, and that in all types of malarial infection studied. Increased values of circulating immunocomplexes had a high frequency rate with all plasmodial species, in P. falciparum infections independently of the degree of parasitemia. (Tab. 7, Ref. 13.)

Pattern of malaria imported by foreign residents under active survey.

An active of imported malaria was carried out on 1432 foreign residents entering Czechoslovakia between 1986-1989. The survey group consisted of adult aged 18-35 years who arrived from malaria endemic regions. Of 660 people surveyed who were from regions of Africa south of the Sahara, 10% were positive for Plasmodium falciparum. Whereas, those arriving from South East Asia had a relatively low prevalence of malaria (2.6%), predominantly P. vivax. Of the 10% of cases testing positive for P. falciparum, 85% had less than 10,000 asexual stages/microliters of blood and 75% were asymptomatic carriers. By contrast, 93.8% of P. vivax/ovale infections were diagnosed because of the onset of symptoms. Both the frequency of seropositivity and the geometrical mean reciprocal titre (IgG), using P. falciparum antigen, were higher in those people arriving from Africa (79% and 1,307) compared with those arriving from S.E. Asia (44.4% and 628). Malaria was confirmed, by blood smear examination, in only 23.8% of the seropositive cohort. There was a positive correlation between the percentage of P. falciparum-positive blood smears and the level of antibody titre. There was no correlation between serum reactivity and level of parasitaemia.

Change of isoenzyme pattern during long-term polyxenic cultivation of Entamoeba histolytica.

Isoenzymes of phosphoglucomutase and hexokinase were repeatedly evaluated using starch gel electrophoresis in polyxenic cultures of Entamoeba histolytica. In two out of 18 strains spontaneous changes of isoenzyme patterns were recorded. While originally they were categorized into virulent group of zymodemes, following isoenzyme analysis classified them as non-virulent. The relation between virulence and isoenzyme pattern is questionable.

Experimental schistosomiasis mansoni: modulation of granulomas by inhibition of collagen cross-link formation. Preliminary report.

beta-Aminopropionitrile (BAPN) is an inhibitor of the lysyl oxidase required for cross-link formation in collagen maturation. The efficacy of BAPN, alone or in association with the anti-schistosomal drug, praziquantel (PZQ), was primarily assessed by measuring the reduction in liver and intestinal egg loads in murine schistosomiasis mansoni. Depending on the treatment group (PZQ, BAPN, BAPN + PZQ), organ-specific effects were observed using microscope image analysis. Most notable was the relatively small size of granulomas in the livers of BAPN-treated mice, which contrasted with the relatively large size and irregular shape of the granulomas in the intestinal tissues of these mice. Mice treated with the combination of BAPN and PZQ had decreased liver and spleen weights, and a significant reduction in the number of eggs trapped in both the liver (86%) and the intestine (99.1%), compared with untreated mice and those given PZQ alone. The lowest number of living eggs/g of tissue in both the liver and intestine was recorded in the combined BAPN + PZQ-treated group. These results suggest that the concurren treatment of infected mice with PZQ and BAPN enhances the release of eggs trapped in the intestine and also results in a significant reduction of liver egg load. The mechanism by which BAPN reduces the number of liver granulomas in PZQ-treated mice is currently being investigated.

Malariometric survey in Keoudom District, Laos: sensitivity of Plasmodium falciparum to anti-malarials and automedication with chloroquine.

A malariometric survey was conducted in the Keoudom District, in the northern part of Vientiane Province, Laos, where an artificial dam-lake on the Nam River is located. The parasite rate of the whole cohort representing 1,105 subjects was 2.44% with the predominance of Plasmodium vivax (70%), while P. falciparum represented 30% with the average parasite density index 3. The low spleen rate (2.3%) characterized the study area as a hypoendemic zone. IFAT antibodies were examined in 419 subjects. The seropositivity of 195 persons < 15 years was 13.7% while in > 15 year old subjects seropositivity was 61.6% with a low GMRT in both groups (140:148). Automedication with aminoquinoline was assayed by urinary analysis in 125 outpatients. Of these, 36 (28.8%) were positive, 89 (71.2%) negative. The frequency of positive blood films for P. falciparum was higher in subjects with aminoquinoline in the urine (36.1%) than in those without (10.1%). Chloroquine sensitivity assay of 15 strains of P. falciparum displayed resistance in 39.3%.

Efficacy of praziquantel treatment of schistosomiasis in a non-endemic country: a follow-up of parasitological, clinical and immunological parameters.

Schistosomiasis patients were immigrants to Czechoslovakia from Angola and Yemen. Most of them had light or moderate infections and felt subjectively healthy. They received treatment with praziquantel (two doses with a total of 40 mg/kg) and were followed up for several years. In nine of 13 patients, Schistosoma haematobium or S. mansoni eggs with undamaged miracidia were detected in biopsies from the bladder or the rectum one year or later after treatment. Granulomatous reactions in the rectum and bladder lesions of stage 1 including thickened bladder walls persisted in most of the patients. Antibody levels against adult S. mansoni worm antigen remained elevated for at least two years after therapy in some patients and declined in others. Among the nine patients, for whom pre- and post-treatment sera were available, the changes in relative levels of antibodies did not strictly correlate with the continued presence of schistosome eggs in, or their absence from, biopsies. We discuss the results obtained with sensitive diagnostic techniques in the absence of subjectively perceived disease.