Epidemiology, clinical manifestations and diagnosis of zoonotic cestode infections: an update.

This paper reviews the literature on zoonotic cestode infections with specific reference to the years 1999-2003. The sources and prevalence of various zoonotic tapeworm infections caused by adult and larval stages of the genera Taenia, Echinococcus, Diphyllobothrium, Hymenolepis and Dipylidium continue to be an important cause of morbidity and mortality, not only in most underdeveloped countries but also in industrialized countries, particularly in rural areas or among immigrant groups from endemic areas. The review gives a detailed report on recent molecular epidemiological studies on the taxonomy and phylogenetic variations in Echinococcus granulosus, immunological tests and imaging techniques used in epidemiological surveys and clinical investigations of important adult and larval tapeworm infections of animals and humans. Larval stages or metacestodes of Taenia solium, Echinococcus spp. and pseudophyllidean tapeworms (Spirometra syn. Diphyllobothrium spp.) may reside in various tissues of their intermediate hosts, including humans. In particular, Cysticercus cellulosae (T. solium) and the larvae of E. granulosus, and E. multilocularis, which are predominantly located in the liver, lungs and central nervous system forming various types of cysts, lead to a complex of systemic diseases such as cysticercosis, cystic echinococcosis and alveolar echinococcosis, respectively. Relatively rare clinical manifestations are seen in the muscles, subcutaneous tissue, spleen, kidneys, bones and body cavities.

Nitroheterocyclic drugs with broad spectrum activity.

The group of biologically active nitroheterocyclic compounds includes various 5- and 2-nitroimidazoles and 5-nitrofurans, which can be used as therapeutic agents against a variety of protozoan and bacterial (anaerobic) infections of humans and animals. The current status in the the treatment of giardiasis, trichomoniasis, balantidiasis, histomoniasis, and amebiasis (including infections due to opportunistic amebas) is presented. The most relevant drugs (benznidazole, furazolidone, metronidazole, misonidazole, nifurtimox, nimorazole, nitazoxanide, ornidazole, secnidazole, and tinidazole) are characterized with regard to their chemical, chemotherapeutic, toxicological, pharmacokinetic, and pharmacological properties, including the mechanism of action and resistance in certain parasitic protozoa.

Antiamoebic activity of 3,3'-fluro-4,4'-di-(pyrrolidine-2-ylidene amino)-diphenyl (liroldine), against experimentally infected intestinal and hepatic amoebiasis.

HL 707, Liroldine, a novel synthetic compound, was found effective against both extraintestinal and intestinal amoebiasis in animal models. Its activity against hepatic infection in golden hamsters is comparable with that of different derivatives of nitroimidazoles used for human treatment. Against intestinal amoebiasis in Wistar rats, the activity was superior to nitroimidazoles and chloroquine. Paramomycin was comparable and diloxanide furoate was marginally superior. The comparative in vitro and in vivo studies with standard marketed drugs and Liroldine indicate an excellent profile of the compound against experimental amoebiasis. LD50 of Liroldine determined in mice is 910 mg/kg x 1, po and 940 mg/kg x 1 ip).

Ultrastructural changes on various Trypanosoma spp. after a 30-year storage period in liquid nitrogen.

Four Trypanosoma species were examined for damage following prolonged storage in liquid nitrogen (-196 degrees C). The stabilates were successfully recovered after a cryopreservation period of approximately 30 years. The structure of specimens was studied by means of light microscopy and scanning (SEM) and transmission (TEM) electron microscopy. All of the species tested--T. evansi, T. equinum, T. brucei, and T. congolense--proved to be infective to mice. However, as compared with controls, the trypomastigote bloodstream forms, which had been frozen and later recovered, showed clear differences. Formerly deep-frozen organisms usually appeared to have shrunk as a result of solution effects, which occur during freezing and thawing. Ultrastructural changes such as separation of the cytoplasm from the pellicle, the occurrence of large vacuoles in the cytoplasm and karyoplasm, a loss of cytoplasmatic ribosomes, membrane injuries, enlargement of the flagellar pocket, and denaturation of chromatin became obvious. The extent of the ultrastructural alterations appeared to be much greater after a cryopreservation period of approximately 30 years than those previously reported after a 13-year storage period. These changes, however, did not result in a complete loss of infectivity to mice.

Treatment of fish parasites. 11. Morphogenesis of Henneguya laterocapsulata Landsberg, 1987 (Myxosporea, Myxozoa), and the effects of a new triazine derivative, HOE 092 V, on its developmental stages: a light and electron microscopy study.

The ultrastructure of sporogenesis was studied in Henneguya laterocapsulata parasitizing the skin of hybrid catfish (Clarias gariepinus x Heterobranchus bidorsalis) in Nigeria. Sporogenesis started when a generative cell was surrounded by a second nondividing cell (i.e., envelope cell). By subsequent divisions of the generative cell, ten cells were produced, which finally became arranged into two spore-producing units. Each unit consisted of a binucleate sporoplasm, two capsulogenic cells, and two valvogenic cells. Apparently capsulogenesis, valvogenesis, and sporoplasm differentiation occurred concomitantly. In research for chemotherapy of fish parasitized by myxosporeans a new triazine derivative, 2-[3,5-alpha-dichloro-4-(4-methyl-sulfonylphenoxy)-phenyl]-1-me thy l- hexahydro-1,2,4-triazine-3,5-dion (HOE 092 V), was tested in vivo against the uni- and multicellular developmental stages of H. laterocapsulata. Naturally infected catfish were incubated in water containing 0, 2.5, 5, and 10 micrograms HOE 092 V/ml or the pure solvent for 3 h. After the fish had been returned into fresh water, they were killed 1 day after the treatment and the plasmodia were studied by means of light and transmission electron microscopy. Starting with a dose of 2.5 micrograms HOE 092 V/ml, the pericyte's outer membrane was broken in the bi- and multicellular stages. The number of ribosomes in the bi- and multicellular stages decreased. In the multicellular stages the rough endoplasmic reticula of the capsulogenic cells were enlarged. Treatment with 5 micrograms HOE 092 V/ml led to breaks in the limiting outer membranes of the capsulogenic cells and to vacuolization of their peripheral cytoplasm. In early prespore stages a decrease in the number of spherical inclusions was recognized.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic N-hydroxylation of diminazene in vitro.

The two N-hydroxylated derivatives (amide oximes) and the corresponding amides of the trypanocidal diamidine diminazene (Berenil, CAS 536-71-0) have been synthesized. These reference compounds made it possible to investigate the in vitro metabolism of the amidine functionalities of diminazene. Diminazene metabolites were detected for the first time, in the form of the corresponding mono- and di(amide oxime), after incubation with 12000 g supernatants from rabbit liver homogenates and careful workup (freeze drying). The identification was based on the behavior in thin-layer chromatography and on comparison of the mass spectral data for the metabolites with those for synthetic material. The N-hydroxylation of diminazene showed the properties typical of a reaction catalyzed by a microsomal monoxygenase. Diminazene amides did not occur as metabolites. The di(amide oxime) of diminazene was tested for its trypanocidal and leishmanicidal activity on various laboratory strains of trypanosomes in mice and on Leishmania donovani in hamsters. The studies showed that the di(amide oxime) has a trypanocidal effect on various strains of trypanosomes (T. brucei, T. vivax, T. congolense and T. evansi), but this is distinctly weaker than that of diminazene. A diminazene-resistant strain of T. rhodesiense is also unaffected by the di(amide oxime). The di(amide oxime) also has a leishmanicidal effect, but this again is distinctly less than that of diminazene.

A modified method for experimental candidosis in mice avoiding lethality.

A model is presented which selected one out of 150 Candida albicans strains for the evaluation of antifungal agents. The mice were inoculated with 6 x 10(5) CFU of strain 352 into the tail vein. The strain has a moderate phospholipase B (PLB) activity in vitro and was originally isolated from a stool sample from a patient in an intensive care unit. This infection leads to very little suffering in the infected animals during the 6-day observation period. Kidney counts at day 5 after infection can give a first indication for a possible fungistatic mechanism. Possible interesting drugs can then be evaluated by a second set of experiments using a longer observation time to investigate the compounds for fungicidal properties. The model suggests that screening for systemic antifungals by avoiding lethality of mice in the first place can be done.

Flow cytometric analysis of Eimeria tenella sporozoite populations exposed to salinomycin sodium in vitro: a comparative study using light and electron microscopy and an in vitro sporozoite invasion-inhibition test.

Eimeria tenella sporozoites exposed to 100, 70, 60 and 50 micrograms salinomycin sodium (SAL)/ml medium 199 at 41 degrees C and then stained with propidium iodide/fluorescein diacetate were analysed by means of flow cytometry (FCM). After 20 min exposure, they showed dose-dependent alterations in their size and shape, i.e. ballooning of most cells, and enhanced intracellular esterase activity as compared with untreated controls. After longer exposure periods (40 and 70 min), inflated cells gradually changed into shrivelled or crumpled, nonviable ones, thereby showing a gradual decrease in esterase activity and a gradual loss of membrane integrity (RFA+). As compared with untreated controls, sporozoites treated with 10 micrograms SAL/ml showed negligible RFA+ values (0.4%-2%), whereas those exposed to 1 and 0.1 microgram SAL ml and to the solvent dimethylsulfoxide (DMSO, 1%) did not, even after 70 min exposure. Slight to severe structural changes manifesting as an extremely wavy surface (1 microgram SAL/ml), vacuolization of the cytoplasm, distension or destruction of the mitochondrion and rupture of cell membranes (10 micrograms SAL/ml) were seen not only at higher SAL concentrations but also (rarely) at lower ones. The ability of sporozoites to invade primary chick-kidney cells was significantly inhibited by 70, 60 and 50 micrograms SAL/ml. In general, there were close relationships between findings obtained using FCM, electron microscopy and an invasion-inhibition test. The results indicate that FCM is a reliable and sensitive technique for characterizing the parasiticidal effects on and the possible mode of action of drugs in free coccidian sporozoites.

Studies for the elucidation of the mode of action of the antimycotic hydroxypyridone compound, rilopirox.

Rilopirox is a synthetic, fungicidal antimycotic agent with hydrophobic characteristics. Its chemical name is 6-[4-(4-chlorophenoxy)-phenoxy-methyl]-1-hydroxy-4-methyl-2-pyridone and it has a molecular weight of 357.79. Rilopirox is very soluble in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) but poorly soluble in water. The amount of antimycotic agent remaining in the solution is dependent on the final concentration of the solvent and the amount of rilopirox used. Complexometric studies show that rilopirox has a high affinity for iron ions [unpubl. data]. Catalase, an iron-containing enzyme, is inhibited by the chelating agent rilopirox. Studies on yeast mitochondria and submitochondrial particles show that rilopirox inhibits the respiratory chain. Complex I (NADH-ubiquinone oxidoreductase) contains iron-sulfur proteins and is the main system which is inhibited.

Secondary metabolites by chemical screening: II. Amycins A and B two novel niphimycin analogs isolated from a high producer strain of elaiophylin and nigericin.

Two novel natural niphimycin analogs, amycins A (5) and B (3) were isolated from the culture broth of the Streptomyces sp. DSM 3816 by chemical screening methods. In addition this strain produces the antibiotics niphimycin (4), elaiophylin (2) and nigericin (1). Fermentation, isolation, structure elucidation and biological activity of the amycins are described.

Rilopirox--a new hydroxypyridone antifungal with fungicidal properties.

Rilopirox, 6-[[p-chlorophenoxy)phenoxy]methyl]-1hydroxy-4-methyl-2(1H)-pyrido ne, is a new fungicidal antimycotic with very low water solubility. It was designed to meet the demand for an intravaginal antifungal with a long skin retention time and a strong killing effect on pathogenic yeasts. In addition, it inhibits all common fungal pathogens in the range 0.98 to 15.6 micrograms/ml. Fungicidal rates vis-à-vis Trichophyton mentagrophytes and Candida albicans under proliferative and non-proliferative conditions are higher than those achieved with common azole and allylamine antifungals. Rilopirox is affected by Fe3+ ions and high concentrations of human serum owing to its chelating activity. The peptone source is of utmost importance to the inhibitory activity of rilopirox whereas the pH value seems to be unimportant so long as it is within the range 5-8.5. Rilopirox appears to meet the demand for an intravaginal antifungal as a result of its very low water solubility of 50 ng/ml and its immediate fungicidal action even under non-proliferative conditions.

Loperamid, an efficacious drug against fish-pathogenic acanthocephalans.

A total of 20 drugs were tested for their efficacy in the treatment of infections involving the two major acanthocephalans infesting rainbow trout in European trout farms. In in vitro experiments, the antidiarrhoeic loperamid and the well-known anthelminthic drug niclosamide showed the best efficacy. Worms treated with loperamid contracted the posterior end of their body, in which severe necrosis of the tegument caused the death of the worms. In in vivo experiments, loperamid was the most efficacious drug: 50 mg/kg given to rainbow trout on 3 consecutive days led to a complete cure. According to preliminary tolerance tests in water baths, the toxicity of this drug is low as compared with that of niclosamide, which is very toxic. Thus, loperamid can be recommended as the drug of choice for therapy of acanthocephalan infections in fish.

Improved techniques for the in vitro cultivation of Eimeria tenella in primary chick kidney cells.

Primary kidney cells of 1- to 4-week-old chickens (PCKC) grown in Flexiperm chambers or culture flasks were infected with ultrapure sporozoites of two Eimeria tenella strains. For the 24-h parasite-free adaptation phase of the PCKC culture, Williams E medium plus 10% foetal calf serum (FCS) was used, and for the subsequent parasite-containing 168-h maintenance phase, we used medium 199 plus 2.5% FCS. Monolayers established during that time enabled the routine development of all schizont generations as well as, in general, young oocysts. The parasite stages propagated in FLEX were rendered visible by modified PAS-AO staining. Sporulated oocytes differed in length and width from those recovered after their passage through chickens. These results show that E. tenella can reliably be reproduced from sporozoites to oocysts in PCKC cultures. However, the yield of oocysts was generally low, indicating that mass production of oocysts is achieved only by passaging sporulated oocysts through chickens.

Increase of DNA content in tissue stages of Entamoeba histolytica strain SFL 3.

The DNA content of culture forms and tissue stages of pathogenic E. histolytica strain SFL 3 were measured photometrically after the nuclei had been stained with the fluorochrome BAO. As a control, the DNA guartity of E. histolytica strain HK 9 and E. invadens were determined by the same method and compared with reference values. Tissue stages were obtained from hamsters experimentally infected by intrahepatic injection of SFL 3 amoebae. Further studies concerning possible changes in the DNA content of tissue stages involved the following methods: (a) isolation of tissue stages from the liver, followed by distinct suspension periods. (b) Infected liver pieces were directly transferred into culture medium; amoebae emigrating therefrom were cultivated. The study demonstrated that tissue stages contained up to 4 times more DNA than did culture forms. After 3 h cultivation, the DNA content of tissue stages decreased to the level of culture forms. Possible reasons for this change are discussed.

Comparative morphology of human and animal malaria parasites. I. Host-parasite interface.

Human and animal malaria parasites (Plasmodium falciparum, P. malariae, P. vivax, P. berghei, P. gallinaceum) were studied using special fixation and standardized methods, with special attention to their effects on host cells. Morphological alterations induced by the parasites in infected erythrocytes included knobs, invaginations, and caveola-vesicle complexes on the surface of the host cell and clefts, microvesicles, and small vesicles in the cytoplasm of the infected erythrocytes. For P. malariae, the ultrastructural study revealed invaginations with associated microvesicles, but knobs did not occur on the surface of infected erythrocytes. The development of invaginations and microvesicles in P. malariae-infected erythrocytes corresponded to the morphological alterations induced by P. vivax. A new hypothesis concerning the origin of Schüffner's dots is discussed.