Immunoblot for the detection of Ascaris suum-specific antibodies in patients with visceral larva migrans (VLM) syndrome.

Visceral larva migrans (VLM) syndrome caused by Toxocara canis larvae was first described in the 1950s. The role of other nematode larvae, i.e. the pig roundworm Ascaris suum as a causative agent of visceral larva migrans-associated symptoms like general malaise, cough, liver dysfunction, hypereosinophilia with hepatomegaly and/or pneumonia, was discussed controversially during the last decades. Recent serological screening studies for specific A. suum antibodies carried out in the Netherlands and Sweden yielded remarkable high seroprevalences, while a number of case reports from Japan report pulmonal, hepatic and cerebral symptoms caused by A. suum larvae after ingestion of infected raw meat (liver) or contaminated vegetables. We present here a sensitive and specific larval excretory-secretory (E/S) antigen-based immunoblot (As-IB) for the serodiagnosis of A. suum-infected patients suffering from symptoms associated to the VLM syndrome. In total, 34 sera from patients with hypereosinophilia and other clinical symptoms associated to the VLM syndrome tested negative for Toxocara sp. antibodies but positive in our newly established As-IB, 30 sera from healthy volunteers, 53 sera from patients with clinically and serologically confirmed toxocarosis and other helminthoses as well as 3 sera from patients with intestinal ascariosis due to Ascaris lumbricoides were included in the study. When evaluated with 30 sera from healthy volunteers and 53 sera from patients suffering from different helminthoses, the calculated specificity of our new As-IB is 95%. Problems hampering the establishment of simple serological screening tests for specific A. suum antibodies, like extensive antigenic similarities between the nematodes Ascaris and Toxocara or the absence of suitable experimental animals, are discussed. We assume that specific serological testing for antibodies of A. suum is very important for the treatment of individual patients on one hand and seroepidemiological investigations will help to clarify routes of transmission on the other hand. Further studies will be necessary to learn more about the extent of A. suum as a causative agent of the VLM syndrome and the role of pigs and their manure as the main source of human Ascaris infections in Austria and other industrialized countries.

Miltefosine and polyhexamethylene biguanide: a new drug combination for the treatment of Acanthamoeba keratitis.

BACKGROUND: In this study, a series of compounds - miltefosine, polyhexamethylene biguanide, chlorhexidine and propamidine isethionate - and combinations of the latter three agents with miltefosine were prepared and used in a rat model for the topical treatment of Acanthamoeba keratitis. METHODS: The corneas of rats were infected with Acanthamoeba hatchetti. On the fifth day, all corneas were microscopically examined in order to determine the grade of infections. Nine groups were then prepared: miltefosine (65.12 µg/mL); chlorhexidine (0.02%); polyhexamethylene biguanide (0.02%), propamidine isethionate (0.1%), miltefosine plus chlorhexidine, miltefosine plus polyhexamethylene biguanide; miltefosine plus propamidine isethionate; infected control; and a non-infected control group. The treatment was continued for 28 days. After the treatment, the corneas were excised and used for Acanthamoeba culture to investigate the presence of Acanthamoeba growth. For the determination of cytotoxicity of the drugs on L929 cells, colorimetric assays were performed. RESULTS: The best treatment results were obtained from the polyhexamethylene biguanide plus miltefosine group; the ratio of fully recovered eyes was 28.4%. It was proven that the miltefosine-polyhexamethylene biguanide combination yielded the highest anti-acanthamoebal activity in that approximately 86% of the eyes were cleared from amoebae. The cytotoxicity values of the miltefosine and the control groups were compared with other groups and found to be statistically different (P < 0.05). CONCLUSION: This in vivo study demonstrates that a miltefosine-polyhexamethylene biguanide combination is highly effective for the treatment of Acanthamoeba keratitis.

Efficacy of miltefosine for topical treatment of Acanthamoeba keratitis in Syrian hamsters.

Acanthamoeba keratitis is a painful corneal infection and difficult to treat because no sufficiently efficient drug has yet been available. The aim of the study therefore was to assess the therapeutic potential of miltefosine on Acanthamoeba keratitis-infected hamster eyes. The cornea of hamsters were infected with Acanthamoeba hatchetti, a human corneal isolate. On the fifth day, all the cornea were microscopically examined in order to determine the degree of infections (G, from 0 to 3). Four groups were then prepared: miltefosine (160 µM); 0.1% propamidine isetionate plus 0.02% polyhexnide; infected control (0.05% ethanol in PBS) and a non-infected control (0.05% ethanol in PBS) groups. The treatment was continued for 28 days. After the treatment, the cornea were excised and used for Acanthamoeba culture to investigate the presence of Acanthamoeba growth. Miltefosine treatment yielded much higher cure scores than propamidine isetionate plus polyhexanide. On the last day of treatment, 85% of the miltefosine-treated eyes were graded as G0; no changes were observed in the uninfected control group eyes; G3 eyes showed only a partial improvement. Furthermore, no Acanthamoeba cells could be recovered from the miltefosine-treated eye samples. Miltefosine appeared to hold necessary therapeutic properties for the treatment of Acanthamoeba keratitis.

Anti-acanthamoeba efficacy and toxicity of miltefosine in an organotypic skin equivalent.

OBJECTIVES: Acanthamoebae can cause infections of several organs, including eye, skin, lung and brain. Except for Acanthamoeba keratitis, these infections are linked to immunodeficiency. Treatment is generally problematic, due to the lack of sufficiently effective and also easily manageable drugs. In a previous study we discovered that miltefosine (hexadecylphosphocholine) is highly active against Acanthamoeba spp. in vitro. The aim of the current study was to evaluate the suitability of miltefosine for the topical treatment of Acanthamoeba infections. METHODS: Storage life and time dependency, susceptibilities of opportunistic bacterial and fungal pathogens, and synergistic and adverse effects of combinations with other anti-Acanthamoeba substances were determined. Moreover, an organotypic skin equivalent was adapted for investigating the penetration of acanthamoebae into the epidermis and the human tissue tolerability of miltefosine. RESULTS: It was shown that miltefosine can be stored as a 2 mM stock solution and also as a 50 microM dilution over a period of 12 months at 4 degrees C without any loss of activity. Efficacies against staphylococci and Candida albicans were established. Acanthamoebae were able to penetrate the skin equivalent within 24 h. This penetration was prevented by treatment with miltefosine, while miltefosine treatment was well tolerated by the skin equivalent. CONCLUSIONS: Miltefosine has been approved for oral and topical treatment of leishmaniasis and may also be a promising candidate for the topical treatment of Acanthamoeba infections.

Activity of methylgerambullin from Glycosmis species (Rutaceae) against Entamoeba histolytica and Giardia duodenalis in vitro.

Entamoeba histolytica and Giardia duodenalis are widespread intestinal protozoan parasites which both spread via cysts that have to be ingested to infect a new host. Their environment, the small intestine for G. duodenalis and the colon for E. histolytica, contains only very limited amounts of oxygen, so both parasites generate energy by fermentation and substrate level phosphorylation rather than by oxidative phosphorylation. They both contain reducing agents able to reduce and activate nitroimidazole drugs such as metronidazole which is the gold standard drug to treat Entamoeba or Giardia infections. Although metronidazole works well in the majority of cases, it has a number of drawbacks. In animal models, the drug has carcinogenic activity, and concerns about a possible teratogenic activity remain. In addition, the treatment of G. duodenalis infections is hampered by emerging metronidazole resistance. Plant-derived drugs play a dominant role in human medicine, therefore we tested the activity of 14 isolated plant compounds belonging to seven different classes in vitro against both parasites. The tests were performed in a new setting in microtiter plates under anaerobic conditions. The compound with the highest activity was methylgerambullin, a sulphur-containing amide found in Glycosmis species of the family Rutaceae with an EC50 of 14.5 µM (6.08 µg/ml) after 24 h treatment for E. histolytica and 14.6 µM (6.14 µg/ml) for G. duodenalis. The compound was successfully synthesised in the laboratory which opens the door for the generation of new derivatives with higher activity.

Comparison of two methods for enumerating circulating tumor cells in carcinoma patients.

BACKGROUND: Monitoring of circulating tumor cells (CTCs) in blood of carcinoma patients treated with novel compounds may be a measurement of treatment effectiveness. Before it can be used clinically, a reliably method is needed to enumerate CTCs. We compared two methods for CTC enumeration, OnkoQuick and the CellSearch system. METHODS: We drew 22.5 ml of blood into three CellSave tubes from 15 healthy donors and 61 patients with metastatic carcinoma. After pooling, 15 ml was processed with OncoQuick and 7.5 ml with CellSearch. RESULTS: With both methods no CTCs were found in healthy donors. At least one CTC was detected in 14 of 61 patients (23%) with OncoQuick and 33 of 61 patients (54%) with CellSearch (P < 0.0001). The number of CTCs detected was larger for CellSearch (mean 20 CTCs/7.5 ml of blood) than for OncoQuick (3 CTCs/7.5 ml; P < 0.0001). CONCLUSION: The CellSearch system is a more accurate and sensitive method to enumerate CTCs. Further studies are warranted to evaluate CTC enumeration by the CellSearch system as a monitoring tool for the evaluation of the efficacy of novel anticancer agents.

In vitro efficacy of curcumin on Trichomonas vaginalis.

Trichomonosis, the disease caused by the protozoan parasite Trichomonas vaginalis, is the most common curable sexually transmitted disease with 174 million cases per year worldwide. The emerging resistance against the current standard therapy with metronidazole is pushing the search for alternative drugs. The purpose of this study was to determine the efficacy of curcumin, a derivate of Curcuma longa, on T. vaginalis. The effective concentrations (ECs) were evaluated using three strains of T. vaginaliswith different metronidazole susceptibilities (ATCC 30001, ATCC 30236 and ATCC 50138) and dilution series of curcumin in 24-well microtitre assays. Curcumin was shown to be highly effective against T. vaginalis, and the susceptibility of the different strains was not affected by an existing resistance to metronidazole. After 24 h of incubation, the EC50 ranged from 73.0 to 105.8 µg/ml and the EC90 from 216.3 to 164.9 µg/ml. In all strains tested, a 100 % eradication of all trichomonal cells within 24 h was reached at a concentration of 400 µg/ml curcumin, the 50-fold concentration still being very well tolerated by human mucosa. Altogether, curcumin seems to be a promising candidate for topical treatment of trichomonosis.

Activity of selected phytochemicals against Plasmodium falciparum.

According to the WHO, in 2008, there were 247 million reported cases of malaria and nearly one million deaths from the disease. Parasite resistance against first-line drugs, including artemisinin and mefloquine, is increasing. In this study the plant-derived compounds aglafolin, rocaglamid, kokusaginine, arborine, arborinine and tuberostemonine were investigated for their anti-plasmodial activity in vitro. Fresh Plasmodium falciparum isolates were taken from patients in the area of Mae Sot, north-western Thailand in 2008 and the inhibition of schizont maturation was determined for the respective compounds. With inhibitory concentrations effecting 50%, 90% and 99% inhibition (IC(50), IC(90) and IC(99)) of 60.95 nM, 854.41 nM and 7351.49 nM, respectively, rocaglamid was the most active of the substances, closely followed by aglafoline with 53.49 nM, 864.55 nM and 8354.20 nM. The activity was significantly below that of artemisinin, but moderately higher than that of quinine. Arborine, arborinine, tuberostemonine and kokusaginine showed only marginal activity against P. falciparum characterized by IC(50) and IC(99) values higher than 350 nM and 180 µM, respectively, and regressions with relatively shallow slopes S>14.38. Analogues of rocaglamid and aglafoline merit further exploration of their anti-plasmodial activity.

Anti-leishmanial activity of plant-derived acridones, flavaglines, and sulfur-containing amides.

Visceral and cutaneous leishmaniases are an important public health problem in endemic geographic regions in 88 countries worldwide, with around 12 million infected people. Treatment options are limited due to toxicity and teratogenicity of the available drugs, response problems in HIV/Leishmania co-infections, and upcoming resistances. In this study, we investigated the anti-leishmanial activity of 13 plant-derived compounds in vitro aiming to find new drug candidates. Toxicity of the compounds was evaluated in human primary hepatocytes, and hemolytic activity was examined in freshly isolated erythrocytes. Two acridones, 5-hydroxynoracronycine and yukocitrine, two flavaglines, aglafoline and rocaglamide, and the sulfur-containing amide methyldambullin showed promising anti-leishmanial activities with 50% effective concentrations (EC50s) of 34.84, 29.76, 7.45, 16.45, and 6.29 µM, respectively. Hepatotoxic activities of 5-hydroxynoracronycine, yukocitrine, and methyldambullin were significantly lower compared to miltefosine and lower or equal compared to artesunate, whereas the ones of rocaglamide and aglafoline were slightly higher compared to miltefosine and significantly higher compared to artesunate. None of the compounds showed hemolytic activity.

High antitrypanosomal activity of plant-derived sulphur-containing amides.

Chagas disease, caused by Trypanosoma cruzi, represents an important public health problem in endemic geographic regions in Middle and South America, affecting 15 million infected people. Treatment options are still limited due to the toxicity of available drugs, parasite resistance and poor drug activity during the chronic phase of the disease. In this study, we investigated the in vitro antitrypanosomal activity of 15 tropical plant-derived compounds with the aim of finding new drug candidates. Three novel sulphur-containing amides (methyldambullin, methylgerambullin and sakambullin) showed promising antitrypanosomal activities, with 50% effective concentrations (EC50 values) after 72 h exposure of 1.7, 1.23 and 5.18 µM, respectively, compared with EC50 values for amphotericin B and benznidazole of 0.71 µM and 30.89 µM, respectively.

Association of autoantibodies against small nuclear ribonucleoproteins (snRNPs) with symptomatic Toxocara canis infestation.

Several studies have demonstrated the occurrence of autoantibodies in the course of infestations with helminth parasites and a number of target proteins have been identified. Sera from patients suffering from toxocarosis, a disease caused by the parasitic roundworm Toxocara canis, and from healthy individuals were tested for autoantibodies by immunofluorescence and immunoblot assays using HEp-2 cells as antigen. A considerable proportion of the sera from patients with toxocarosis-associated symptoms were autoantibody-positive, with a speckled staining pattern in the immunofluorescence test (62%) and with anti-snRNP reactivity in the immunoblot assay (98%). In contrast, significantly fewer sera from asymptomatic individuals scored positive in these assays (18% in the immunofluorescence test, P < 0.005; 24% in the immunoblot, P < 0.005). Although the causative link between Toxocara infestation and the occurrence of autoantibodies is still unclear, our results show that increased amounts of autoantibodies are associated with clinical symptoms of inflammation. Thus a serum test for autoantibodies in toxocarosis patients might be a valuable gatekeeper assay for the decision for or against anti-inflammatory treatment.

Cytotoxic activities of alkylphosphocholines against clinical isolates of Acanthamoeba spp.

Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 microM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.