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20 février 2011 7 20 /02 /février /2011 10:42
Exp Parasitol () (2010)
Toxocara canis: potential activity of natural products against second stage larvae in vitro and in vivo.
Unidade de Helmintologia e Malacologia Médicas/ UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
ABSTRACT

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox(R) was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox(R) was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox(R), the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17(th) day post infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo. Copyright © 2010. Published by Elsevier Inc. DOI: 10.1016/j.exppara.2010.04.023 
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NUTRIDESINTOX (Chenopodium ambrosioides and Pycnanthus angolensis)

 

Title: In vitro and in vivo anthelmintic activity of plant extracts and a nutritional supplement against Toxocara canis

Authors:

Mariana Reisa, Maria J. Ferreirab, Ana R. Monsalve-Puelloc, Miguel Correiad, Alcione Trincaa, Maria A.A. Grácioa

 

Address:

a Unidade de Helmintologia e Malacologia Médicas/ UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal

b iMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal

c Nutrialimentos Jesana S.L., Barcelona, Espanha

d Departamento de Histologia, Embriologia e Biologia Celular, Faculdade de Ciências Médicas Universidade Nova de Lisboa, Lisboa, Portugal

 

Abstract:

The aim of this study was to search for new treatments for human toxocariasis using in vitro and in vivo experiments and ethnobotanical data. Thus, extracts from two plants (Chenopodium ambrosioides and Pycnanthus angolensis) used in folk medicine as anthelmintics, and a nutritional supplement (Nutridesintox®) have been evaluated against Toxocara canis larvae. Albendazole was used as a standard reference. In the in vitro experiments, the nutritional supplement Nutridesintox® was the most effective, followed by the hexane and the dichloromethane extracts and the infusion of C. ambrosioides.

The methanolic, ethanolic and the methylene chloride extracts of P. angolensis showed no anthelmintic efficacy. Due to their in vitro anthelmintic activity, the nutritional supplement, the hexane extract and the infusion of C. ambrosioides were selected for the in vivo experiments.

These products were administered orally to T. canis-infected CD1 mice in single doses during three consecutive days, commenced on day 10 post-infection. The efficacy of treatment was evaluated on day 5 after the last dose, by counting the numbers of T. canis larvae in the brain, liver, lungs and muscles, detection of IgM and IgG antibodies by ELISA and histological analysis of liver and lungs. The dynamics of the antibodies on the treated groups showed a similar profile as compared to the control group, statistically significant differences were not found. The different treatments did not show a clear evidence of reduction of larvae burden in the infected tissues. The level of infection in the brain was higher than in the other organs examined, except for the group treated with the infusion of C. ambrosioides.

Interestingly, a reduction of inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. The in vitro and in vivo results call for further studies on these natural products, in order to find alternative therapeutic regimens for human toxocariasis.

 

Keywords: Anthelmintic activity; Chenopodium ambrosioides; Pycnanthus angolensis Toxocara canis

 

1. Introduction

 Toxocara canis(Werner, 1782), the roundworm of dogs, is the etiologic agent of human toxocariasis. As the dog constitutes one of our most common pets, this ensured the worldwide distribution of this zoonotic disease. Commonly the infection is acquired after ingestion of embryonated T. canis eggs that can be present in soil contaminated with dog faeces. Children are usually the most susceptible to infect because of their habits of pica (Despommier, 2003). As larvae hatch in the stomach they penetrate the mucosal epithelium and thereafter remain developmentally arrested in the tissue phase. Although they do not grow or differentiate in the paratenic host they maintain an active metabolism and migratory behaviour (Maizels et al., 2000). Their wandering around the body gives rise to two main syndromes; visceral larva migrans (VLM), in which the major organs are affected and ocular larva migrans (OLM), when it affects the eye, in some cases, it can lead to unilateral blindness (Magnaval et al., 2001)

 The most common anthelmintic drugs used for the treatment of toxocariasis belong to the benzimidazole carbamates group (Pawlowski, 2001). However, these drugs have a low bioavailability for tissues, due to their extremely low solubility, resulting in relatively high doses over a long periods of time (Hrčkova and Velebný, 2001). In addition, the use of these drugs for treatment of other helminths leads to drug resistance, as it was already verified in Mali for Necator americanus (De Clercq et al., 1997). Thus, new drugs for treatment of helminthic diseases are urgently needed. The study of plants used in traditional medicine as anthelmintics could give new insights for active compounds. The co-evolution human-helminth has a long existence and the use of plants for treatment of helminth parasites is recorded since the prehistorical times (Reinhard et al., 1985). Since then, the popular knowledge of medicinal plants has been consolidated throughout generations by trial and error experiences.

Chenopodium ambrosioides L. (Chenopodiaceae) is a species originally from Central and South America, which grows wild in Portugal and in the Mediterranean region. In Portugal is popularly known as “formigueira” or “erva-de-Santa-Maria”, the infusions made of leaves and inflorescences are used as vermifuge and for the treatment of asthmatic and nervous manifestations (Tecedeiro, 1996).

Pycnanthus angolensis(Welw.) Warb. (Myristicaceae), is known as “pó-casson” in the islands of São Tomé e Principe. Preparations of its stem bark are used of as anthelmintic, analgesic, haemostatic and for the treatment of pneumonial infections (Betti, 2002; Diehl et al., 2004).

Nutridesintox® is a nutritional supplement composed of fruits, vegetables and seeds, designed to purify the organism from toxins produced by the metabolic activity of parasites.

The objective of the current study was to assay some plant extracts from Chenopodium ambrosioides and Pycnanthus angolensis and one nutritional supplement (Nutridesintox®) against T. canis larvae in order to evaluate the potential in vitro and in vivo anthelmintic activity.

 

2. Materials and Methods

2.2 Parasite

Toxocara canis adult worms were collected from naturally infected puppies by using an anthelmintic (pyrantel pamoate, Pfizer). The eggs were removed from the worm uterus and were maintained in 1% formalin at 27 ºC until the development of the infective stage.

 

2.1 Plant material and nutritional supplement preparations

Chenopodium ambrosioides was collected in Portugal (authenticated by voucher number). The plant extracts were obtained by sequentially extracting 50 g of air-dried powdered plant material with 500 ml of hexane, dichloromethane, ethyl acetate and methanol for 48 h at room temperature. After filtration, the extracts were concentrated to dryness, under reduced pressure at 40-45 ºC, using a Büchi rotatory evaporator, and then stored at 4ºC until used.

This species was also obtained in a traditional medicinal herb shop and was authenticated using Franco (1971). An infusion was prepared with this plant by adding 100 ml of boiling water to 10 g of the dried aerial parts and leaving it standing for 10 min. The infusion was sterilized through a 0.22 µm filter and stored at -20 ºC.

The stem bark of Pycnanthus angolensis was collected in São Tomé and Príncipe islands and extraction procedures were previously described in Abrantes et al (2008).

Nutridesintox® was obtained from Nutrialimentos Jesana S.L., Barcelona (http://www.nutricioncuantica.com). This nutritional supplement is composed by carrot, pumpkin, garlic, sesame and wheat. One capsule (725 mg) was dissolved in 2.5 ml of distilled water for 1 hour at 37 ºC and then centrifuged at 4000 rpm for 10 min. The supernatant was used directly in the in vitro experiments.

 

2.3 In vitro experiments

2.3.1. Sample preparation

Hexane and dicloromethane (DCM) extracts of C. ambrosioides; DCM, ethanolic (EtOH) and methanolic (MeOH) extracts of P. angolensis were diluted in dimethylsulphoxide (DMSO) to obtain the following concentrations 0.01; 0.05 and 0.1 mg/ml.

Albendazole (Zentel, GlaxoSmithKline) was also diluted in DMSO to obtain the same concentrations.

Infusion of C. ambrosioides and the aqueous extract of Nutridesintox® were diluted in Hank’s Balanced Salts Solution (HBSS) (Sigma-Aldrich) to obtain the concentrations of 25, 50 and 100 µl/ml.

2.3.1 Nematocidal activity test on T. canis larvae

Toxocara canissecond-stage larvae were hatched according to De Savigny (1975). These larvae were maintained in HBSS medium at 37 ºC with an atmosphere of 5 % CO2. The in vitro test was preformed in 24-well microplates (30 larvae/well) with the test substances, albendazole as standard anthelmintic and HBSS medium and DMSO as solvent controls. All assays were preformed in duplicate. After 48 h of exposure the mobility of the larvae were examined using an inverted microscope. Nematocidal activity was evaluated in terms of relative mobility (RM). This method was originally developed by Kiuchi et al. (1987), however, due to the different types of movements observed during the assays, we had to adapt this system, by adding more scores as it is shown in Table 1.

 

2.4. In vivo Experiments

2.4.1 Animal infection

Male CD-1 mice with 8-week-old, weighing 35 g were obtained from the animal facilities of the Biotério do Instituto de Higiene e Medicina Tropical, and maintained under standard laboratory conditions, according to the European Union requirements (86/609/CEE). Mice were infected with 300 embryonated eggs by gastric intubation via a metal cannula coated with silicon. Seven mice were used in each experimental group.

 

2.4.2. Animal treatment

The test substances were selected based on the in vitro test results. All the treatments were administered daily from day 10 post-infection (p.i) to 12 p.i.. The extracts of C. ambrosioides and albendazole were administered orally using the following doses: hexane extract of C. ambrosioides (30 mg/Kg/ 0.1 ml); infusion of C. ambrosioides (15 ml/Kg/0.5 ml) and albendazole (800 mg/Kg/0.1 ml). For the nutritional supplement administration, mice were separated into individual cages with a mixture of standard food and the content of two capsules of Nutridesintox® per day.

All mice were sacrificed at 17th day p.i..

 

2.4.3. Larvae recovery from infected tissues

The brain, liver, lung and musculature were finely minced and incubated in a digestion fluid (pepsin 0.25 g, HCl 1 ml, H2O 100 ml) during 24 h at 37 ºC. The brain was incubated separately from the other organs. Larvae from the sediment were counted under a microscope.

 

2.4.4. ELISA test for determination of IgM and IgG antibodies in the serum

Blood was collected from the tail of mice from the different groups before the infection, before the treatment and on the 17thday p.i..

Toxocara canis excretion-secretion (TES) antigen was prepared according to De Savigny (1975).

The enzyme-linked immunosorbent assay (ELISA) for detection of IgM and IgG was performed in 96-well maxi-sorb plates (Nalgene Nunc). Plates were sensitised with 10 μl/ml of TES diluted in 0,1 M sodium carbonate buffer ( pH 9.6; 100 µl/well) and incubated for 30 min at 37 ºC, followed by overnight incubation at 4 ºC. Plates were washed three times with phosphate-buffered saline (pH 7.2) containing 0.05% v/v Tween-20 (PBS/Tween). Plates were blocked with a solution of 1 % bovine serum albumin during 1h 30 min at room temperature; plates were then washed three times with PBS/Tween.

Serum samples were used at a dilution of 1:400 in PBS/Tween. After incubating 1 h at room temperature, plates were washed and 100 µl of anti-mouse IgM or IgG conjugated to alkaline phosphatase (Sigma-Aldrich, USA) at a dilution of 1: 10.000 in PBS/Tween were added for 1h 30 min at room temperature. After the plates were washed, substrate (p-nitrophenyl phosphate, Sigma-Aldrich) was added to each well and the reaction was stopped by adding 3N NaOH. The optical density values were measured at 405 nm.

 

2.4.5. Histology

Livers and lungs of mice from the experimental groups were removed on 17thday p. i.. Tissues were fixed in 10 % formalin and embedded in paraffin blocks. Sections of 5 µm of each organ were stained with hematoxylin-eosin (HE). Analysis of the sections was performed in a blinded fashion.

 

2.4.6. Statistical analysis

Statistical differences on the larvae burden between the groups were determined using Wilcoxon-Mann-Whitney test. The production of IgM and IgG antibodies between the groups was statistically tested using Kruskal-Wallis test, a Spearman’s correlation was also preformed. A probability value p < 0.005 was considered statistically significant. Analysis was performed using SPSS 16.0 statistical package.

 

Results

3.1. In vitro experiment

Albendazole, extracts of C. ambrosioides, extracts of P. angolensis and an aqueous extract of Nutridesintox® were evaluated for their in vitro anthelmintic activity against T. canis second-stage larvae. The results of the nematocidal activity are presented in Table 2. In the negative control assays (HBSS medium and DMSO) a relative mobility of 100 % was measured, which indicates that these substances did not have any anthelmintic effect. At the highest concentration tested, except for the DCM extract of P. angolensis, all the other tested extracts had higher nematocidal activity than albendazole (Table 2). The aqueous extract of Nutridesintox® exerted the highest nematocidal activity, with 65 % of dead larvae (Fig. 1). All the extracts of C. ambrosioides showed similar relative mobility values at the highest concentration tested. Interestingly, the hexane extract of C. ambrosioides was the only that exhibited nematocidal effect even at the lowest concentration. The extracts of P. angolensis showed a low anthelmintic effect (Table 2).

Thus, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were chosen for the in vivo study.

 

3.2. In vivo experiment

The effect of the treatments was evaluated on the day 5 post-therapy. At this day, the accumulation of larvae in the brain was higher than in the other organs studied, except for the group treated with infusion of C. ambrosioides (Table 3). The groups treated respectively with albendazole, infusion of C. ambrosioides and with Nutridesintox® showed significant differences between larval numbers in the studied organs in comparison with the control (Table 3). However, these results did not show a reduction of larvae burden post therapy.

The levels of IgM and IgG antibodies in the serum of mice from control and treated groups are shown in Fig. 2. Using the non-parametric Kruskal-Wallis test, no significant differences in the levels of IgM (χ2= 4,048; df = 4; p = 0,399) and IgG (χ2= 9,202; df = 4; p = 0,056) between the different treatments were found. In addition, the analysis with the non-parametric Spearman correlation shows a non-linear association between the IgM levels and the number of larvae (Rs=-0,200; p=0,747) and the same was verified for the IgG levels and the number of larvae (Rs=0,100; p= 0,873), which may show a certain independence of these two variables.

The livers and lungs from mice of all experimental groups were analysed by histology. The uninfected controls that received treatment (albendazole, hexane extract and infusion of C. ambrosioides and Nutridesintox®) showed a normal hepatic and pulmonary structure and did not exhibit any observable signs of toxicity (data not shown). The liver sections of T.canis-infected mice treated with the infusion of C. ambrosioides and Nutridesintox® showed the typical inflammatory reaction produced by the migrating larvae, in comparison to the control this reaction seems to be more moderate (Fig. 3 B, D and F). The infected mice treated with albendazole and with the hexane extract of C. ambrosioides did not show signs of inflammatory infiltrates (Fig. 3 C and E).

Regarding to the lung sections, the effects of the treatments were not so evident, as it was observed the presence of inflammatory infiltrates in all experimental groups (Fig. 4). However, a slightly inflammatory reaction was observed for the group treated with the hexane extract (Fig. 4 E).

 

Discussion

Medicinal plants and other natural products have long been used in folk medicine. In the developed countries the depopulation of rural areas, led to the loss these ancient practises. Nowadays, there is an increasing interest in natural products so as to have alternatives to conventional medicine and for the development of new drugs. In the present study we have investigated the anthelmintic activity of some plant extracts and a nutritional supplement against T. canis larvae.

Although, there is no consensual drug for the treatment of visceral larva migrans caused by T. canis, albendazol is the most commonly used drug (Sturchler et al., 1989; Magnaval et al., 2001; Pawlowski, 2001). However, it seems to have a weak action against the T. canis larvae in vitro, as it was demonstrated on our in vitro assays as well as in other studies (Satou et al., 2005; Márquez-Navarro et al., 2008).

From all the natural products tested the nutritional supplement was the one that showed the highest anthelmintic activity in vitro. This supplement is composed of carrot, garlic, pumpkin, sesame and wheat bran that are used in folk medicine as anthelmintics (Elisha et al.,1987; Waller et al., 2001; Salgueiro, 2005 Cunha et al., 2006). A synergistic effect between the components of this supplement might have been the responsible for the high anthelmintic activity observed in vitro. However more studies should be addressed in order to understand this in more detail.

The in vitro assay of the C. ambrosioides extracts showed interesting results.It is long known that the anthelmintic properties of this species are due to the monoterpene  ascaridole (Nelson, 1920). The hexane and DCM extracts, which are rich in this compound, showed an anthelmintic activity similar to the infusion of C. ambrosioides. Some authors suggest that the anthelmintic activity of the aqueous infusions of this species is due to more hydrophilic compounds and not to ascaridole (MacDonald et al., 2004; Gadano et al., 2006). MacDonald et al. (2004) verified that ascaridole-free infusions of C. ambrosioides retained the anthelmintic properties against Caenorhabditis elegans.

Even though the extracts of P. angolensis did not show anthelmintic activity they cannot be considered to be completely inactive. These results could have been due to the concentrations tested as the methanolic and ethanolic extracts showed to have a higher activity compared to albendazole. For this species other authors observed that ethanolic extracts had anthelmintic activity against Haemonchus contortus (Diehl et al., 2004) and antimalarial activity against Plasmodium falciparum (do Céu de Madureira et al., 2002). Methanolic extracts were shown to have anthelmintic activity against Eudrilus eugeniae (Gbolade & Adeyemi, 2008) and leishmanicidal activity against Leishmania major (Onocha et al., 2008).

Regarding to the in vivo evaluation the tested substances were not particularly effective. The brain was the most parasitized organ on the 17thday p. i., which indicates that the migratory larvae were in myotropic-neurotropic phase. Horiuchi et al. (2005) administrated albendazole (100 mg/kg) between the 13thto the 21st day p.i. and did not find significant differences between the numbers of larvae recovered from the brain in comparison to the control. Abo-Shehada & Herbert (1984) verified that the dose of 100 mg/kg of albendazole administrated on the 10th-13thday p.i. did not have anthelmintic efficacy. These authors suggested that larvae in this migratory phase are less susceptible to drug actions than those in hepato-pulmonary phase.

In the groups treated respectively with albendazole, infusion of C. ambrosioides and Nutridesintox® the number of recovered larvae was significantly higher than the number of recovered larvae from the control. Lescano et al. (2004) observed that in BALB/c mice treated with cyclosporine A (immunosuppressant drug) the burden of T. canis larvae was higher when compared to the control and that there was a delay in IgG production. One explanation to our results could be that these substances had exerted an immunosuppressant effect, therefore reducing the natural capacity of elimination of parasites. However, up to the fifth day post-treatment there were no significant differences in IgM and IgG production between the groups.

Interestingly, despite the fact that in the group treated with the hexane extract of C. ambrosioides a reduction of the larvae burden has not been verified; it was observed an absence of inflammatory infiltrates in the liver sections and a reduction of the inflammatory infiltrates in the lungs. In the group treated with albendazole an absence of inflammatory infiltrates in the liver sections was also observed. This could be related to the fact that albendazole is metabolized in the liver to albendazole sulfoxide, which has an anthelmintic effect (Lacey, 1990).

The present results showed a correlation between the in vitro and in vivo experiments and ethnobotanical data, namely to the hexane extract of C. ambrosioides. This study also highlights for the necessity of comparison of the two phases of larval migration and to have a multifactorial analysis (larvae counts, immunology, histology) when planning any search for new drugs for use in the treatment of toxocariasis.

 

Table 1. Criteria for evaluating the effect of drugs on Toxocara canis larvae

State of larva

Score (n)

Fast movement using the whole body

5

Intermediated movement using the whole body

4

Slow movement using the whole body

3

Moving with only a part of the body during the observation

2

Immobile but not dead

1

Dead

0

Motility index (MI) = ∑n Nn/ ∑Nn, where Nn : number of larvae with the score of n.

Relative mobility (RM) = MIsample/ MIcontrolx 100

 

 

Table 2. In vitro anthelminthic activity against Toxocara canis second-stage larvae.

Test substance

Relative Mobility (%)

 

 

Concentration (µg/ml)

Concentration (µl/ml)

 

0,01

0,05

0,1

25

50

100

 

Albendazole

91

77

77

 

 

 

 

C. ambrosioides - hexane

33

21

20

 

 

 

 

C. ambrosioides - DCM

72

30

22

 

 

 

 

C. ambrosioides – infusion

 

 

 

52

38

24

 

P. angolensis - DCM

71

98

85

 

 

 

 

P. angolensis - EtOH

72

62

53

 

 

 

 

P. angolensis - MeOH

95

67

42

 

 

 

 

Nutridesintox®

 

 

 

76

44

15

 

DCM: dicloromethane; EtOH: ethanol; MeOH: methanol

 

 

 

 

 

 

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Parasitol Res. 2010 Nov 26. [Epub ahead of print]

The effects of different plant extracts on nematodes.

Klimpel SAbdel-Ghaffar FAl-Rasheid KAAksu GFischer KStrassen BMehlhorn H.

Biodiversity and Climate Change Center (BiK-F), Goethe-University Frankfurt, Frankfurt, 60325, Germany.

Abstract

The anthelminthic efficacy of some differently obtained extracts of several plants was tested in vivo in laboratory animals and in vitro. The extracts were obtained by ethanolic, methanolic, aqueous, or chloroform, respectively, acetonitrile polyethylenglycol (PEG) and/or propylencarbonate (PC) elution at room temperature or at 37°C. The plants used were bulbs of onions, garlic, chives, coconut, birch tree, ananas, cistrose, banana, chicory, date palm fruit, fig, pumpkin, and neem tree seeds. The worm systems tested both in vivo and in vitro were Trichuris muris and Angiostrongylus cantonensis but only in vivo Toxocara cati. The tests clearly showed that the different extraction methods eluted different components and different mass amounts, which had different efficacies against the above-cited worms. In vitro effects against A. cantonensis and T.muris were best with aqueous extracts, followed by chloroform extracts. The other plant extracts showed only low or no effects on A. cantonensis in vitro. In the case of T. muris, best results were obtained in vivo and in vitro with PEG/PC extracts of the onion followed by the aqueous extract of coconut. The complete elimination of worms in the in vivo experiments with T. muris was obtained when infected mice were treated with a 1:1 mixture of extracts of coconut and onion being produced by elutions with a mixture of 1:1 PEG and PC and fed daily for 8 days. T. cati in a naturally infected cat was eliminated by daily oral application of 6 ml coco's fluid for 5 days. This study shows that a broad spectrum of plants has anti-nematodal activities, the intensity of which, however, depends on the mode of extraction. This implicates that, if results should be really comparable, the same extraction methods at the same temperatures have to be used. Furthermore, efficacy in in vitro systems does not guarantee as good-if at all-efficacy in vivo.

PMID: 21110041 [PubMed - as supplied by publisher]

 

 

Exp Parasitol. 2010 Oct;126(2):191-7. Epub 2010 May 4.

Toxocara canis: potential activity of natural products against second-stage larvae in vitro and in vivo.

Reis MTrinca AFerreira MJMonsalve-Puello ARGrácio MA.

Unidade de Helmintologia e Malacologia Médicas/UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal. mariana.a.reis@gmail.com

Abstract

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.

Copyright 2010 Elsevier Inc. All rights reserved.

PMID: 20447397 [PubMed - indexed for MEDLINE]

 


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(Il existe un séro diagnostic) 

 

Rev Peru Med Exp Salud Publica. 2010 Dec;27(4):613-620.

[Diagnosis of human toxocarosis.]

[Article in Spanish]

Roldán WHEspinoza YAHuapaya PEJiménez S.

Facultad de Medicina, Universidad Nacional Mayor de San Marcos, Lima, Perú

Abstract

Human toxocarosis is an important parasitic zoonosis caused by larval stages of Toxocara species, the roundworms from dogs and cats. Larval migration through different soft tissues in the human generates several clinical entities in the patient, such as visceral larva migrans, ocular toxocarosis, and neurotoxocarosis. Definitive diagnosis by histopathological methods is very difficult or almost impossible and, nowadays, the diagnosis is usually made by clinical signs/symptoms, epidemiological background of the patient and the use of hematological and immunological tests which finally help to confirm the clinical suspicion of the illness. The purpose of this paper was to update the available knowledge on the use of different tools for both the diagnosis and following up of human toxocarosis.

PMID: 21308204 [PubMed - as supplied by publisher]

 

 

Ugeskr Laeger. 2011 Jan 17;173(3):186-9.

[Toxocariasis].

[Article in Danish]

Stensvold CRNielsen HVPetersen E.

Afdeling for Bakteriologi, Mykologi og Parasitologi, Statens Serum Institut, Artillerivej 5, 2300 København S, Denmark. RUN@ssi.dk

Abstract

The clinical presentation of toxocariasis, a zoonotic parasitosis transmitted from dogs and cats to humans, can be very diverse, which is one of the reasons why Toxocara-related disease may go unnoticed. This paper gives a brief summary of the various clinical presentations (covert/common toxocariasis, visceral larva migrans, ocular toxocariasis and neurotoxocariasis), diagnostic and differential-diagnostic considerations as well as treatment and prevention. In brief, the diagnosis of human toxocariasis relies mainly on patient data, anamnestic information, symptoms, eosinophil count and total-IgE levels.

PMID: 21241625 [PubMed - indexed for MEDLINE]

 

Wiad Parazytol. 2010;56(2):117-24.

[The spread of nematodes from Toxocara genus in the world].

Borecka A.

Instytut Parazytologii im. W. Stefańskiego, Polska Akademia Nauk, ul. Twarda 51/55, 00-818, Warszawa. ab@twarda.pan.pl

Abstract

Twenty seven species belong to the genus Toxocara. Most of the species infect Carnivora from families: Canidae, Felidae, Viverridae, Procyonidae, Mustelidae and Herpestidae. The most widespread species are: T. canis, T. cati and T. vitulorum. The life cycle of Toxocara spp. is connected with young animals and adults with the lowered immune response. Three of the Toxocara species: T. canis, T. cati and T. pteropodis are the aetiological agents of human toxocariasis.

PMID: 20707295 [PubMed - indexed for MEDLINE]

 


 

 

Allergy Asthma Immunol Res. 2010 Oct;2(4):267-70. Epub 2010 Aug 20.

A case of recurrent toxocariasis presenting with urticaria.

Kim MHJung JWKwon JWKim TWKim SHCho SHMin KUKim YYChang YS.

Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.

Abstract

Human toxocariasis is the most prevalent helminthiasis in Korea and other industrialized countries. The clinical features of toxocariasis are diverse, according to the involved organ. Typically, Toxocara spp. infection is easily treated with 400 mg albendazole twice a day for 5 days. However, we experienced a case of recurrent toxocariasis that was refractory to this standard therapy and presented with urticaria, an uncommon symptom in toxocariasis. A 35-year-old male visited our emergency room because of abdominal pain. He had recently consumed raw cow liver (3 weeks prior to presentation). Laboratory analyses revealed eosinophilia (1,612 cells/µL) and increased total IgE (3,060 IU/mL). Chest X-ray showed multiple lung nodules in both lungs, and computed tomography revealed multiple ground-glass opacities in both lungs and multiple tiny liver abscesses. Liver biopsy revealed an eosinophilic abscess. Enzyme-linked immunosorbent assay findings for Toxocara antigens were positive (optical density, 2.140), leading to a diagnosis of toxocariasis. We initiated a 5-day treatment with albendazole and prednisolone; however, 6 days after completing the treatment, the patient again experienced urticaria and severe itching that could not be controlled by antihistamines or hydrocortisone cream. A second bout of eosinophilia suggested recurring toxocariasis, for which we prescribed a second round of albendazole. Despite an initial improvement in his symptoms, the patient returned after 6 weeks complaining of abdominal pain for 6 hours, which was reminiscent of his first attack; he also exhibited eosinophilia. Accordingly, albendazole was administered once more for an additional 3 weeks, and his symptoms resolved.

 

 


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La colonisation de l'intestin par  trichuris trichiura améliore la production de mucus via l'amélioration des cytokines TH2 et IL-22 dans la colite ulcérative.

 

 

 

Sci Transl Med. 2010 Dec 1;2(60):60ra88.

 

IL-22+ CD4+ T cells are associated with therapeutic trichuris trichiura infection in an ulcerative colitis patient.

 

Broadhurst MJLeung JMKashyap VMcCune JMMahadevan UMcKerrow JHLoke P.

Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, CA 94143, USA.

Abstract

Ulcerative colitis, a type of inflammatory bowel disease, is less common in countries endemic for helminth infections, suggesting that helminth colonization may have the potential to regulate intestinal inflammation in inflammatory bowel diseases. Indeed, therapeutic effects of experimental helminth infection have been reported in both animal models and clinical trials. Here, we provide a comprehensive cellular and molecular portrait of dynamic changes in the intestinal mucosa of an individual who infected himself with Trichuris trichiura to treat his symptoms of ulcerative colitis. Tissue with active colitis had a prominent population of mucosal T helper (T(H)) cells that produced the inflammatory cytokine interleukin-17 (IL-17) but not IL-22, a cytokine involved in mucosal healing. After helminth exposure, the disease went into remission, and IL-22-producing T(H) cells accumulated in the mucosa. Genes involved in carbohydrate and lipid metabolism were up-regulated in helminth-colonized tissue, whereas tissues with active colitis showed up-regulation of proinflammatory genes such as IL-17, IL-13RA2, and CHI3L1. Therefore, T. trichiura colonization of the intestine may reduce symptomatic colitis by promoting goblet cell hyperplasia and mucus production through T(H)2 cytokines and IL-22. Improved understanding of the physiological effects of helminth infection may lead to new therapies for inflammatory bowel diseases.

PMID: 21123809 [PubMed - in process]

 


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Helminths could suppress immune disorders by promoting healthy mucus production in the intestine

 

Scientific American : 

http://www.scientificamerican.com/article.cfm?id=helminthic-therapy-mucus

 

In 2007, parasite immunologist P'ng Lokesat down for lunch at a University of California, San Francisco, cafeteria with an inquisitive man who had called him earlier that week. Their chosen topic of conversation would deprive many people of an appetite, but the scientist and his guest shared an intellectual hunger for a stomach-churning subject: gut worms—specifically, tiny worm-like parasitic organisms called helminths that live nestled in the gastrointestinal tracts of their hosts.

Loke was fully prepared to answer the man's questions about the parasites he knew so well, but what he did not realize was that his companion had more than just questions—he had worms burrowed in his intestinal walls, worms he had deliberately swallowed. Together, Loke and the worm-wrangler embarked on a research project, the results of which appear today in the December 2010 issue of Science Translational Medicine.

The 35-year-old man who had lunch with Loke was quite healthy in 2007. But only a few years earlier he was in the throes of an inflammatory bowel disease known asulcerative colitis. An autoimmune disease, ulcerative colitis inflames the colon and leaves it rife with open sores; patients experience intense abdominal pain, vomiting, diarrhea, rectal bleeding and weight loss. While searching for treatments, the man discovered the work of Joel Weinstock, a gastroenterologist, parasitologist and immunologist at Tufts University who has pioneered research on helminthic therapy—treating autoimmune diseases by deliberately infesting patients with parasitic worms, such as whipworm and hookworm. 

The results of Loke's new case study—the most recent of only five studies that investigate helminthic therapy in people instead of animals—suggest that helminths may ease the symptoms of autoimmune diseases by increasing mucus production. 

"It's a unique study—there's nothing like it before," says Weinstock, who was not involved in the new research. "In this case they had a very unique patient—one who was self-infecting with helminths." Clinical trials on helminthic therapy are particularly difficult to arrange because helminths are live pathogens and have not been officially approved as therapeutic agents by any governmental agency, although the U.S. Food and Drug Administration has granted pig whipworm (Trichuris suis) the status of Investigational New Drug. In contrast to human whipworm (Trichuris trichiura), the porcine variety cannot survive inside the human gut for very long.

"The researchers noticed a specific pattern of behavior, cycling between remission and active disease depending on when the patient infected himself with helminths," Weinstock adds. "This is not a double-blind study, but the pattern is highly suggestive that the worms helped this patient. The major point of this paper is the potential mechanism—mucus production—which has not been looked at properly before."

The Might of Mucus

In the new study, Loke—who is now with New York University—analyzed the man's medical records prior to 2007 and personally tracked the man's health from 2007 onwards. In 2004 the man swallowed a vial of salty liquid brimming with 500 human whipworm eggs, which he obtained from a parasitologist in Thailand. Three months later, he slurped down another 1,000 eggs. The larvae hatched and matured within his gastrointestinal tract, burying their heads in the intestinal wall. By mid-2005, he was virtually symptom free and required no medical treatment for his colitis, except occasional anti-inflammatory drugs to suppress flare-ups. The nearly complete dismissal of colitis symptoms is especially striking because human whipworm infection can itself cause digestive problems, including diarrhea, abdominal pain, nausea, vomiting and, in extreme cases, rectal prolapse. Severe infections can also cause anemia and stunt the growth of children. 

In 2008, the number of whipworm eggs in the man's stool began to dwindle, dropping from more than 15,000 per gram to fewer than 7,000 per gram. As the eggs disappeared, the symptoms of colitis returned. So the man infected himself with another 2,000 whipworm eggs and, a few months later, his symptoms practically vanished once again. Repeated colonoscopies revealed that wherever worms colonized his colon, the symptoms of colitis were significantly reduced or nonexistent. 

During the 2008 relapse, the researchers found that immune cells in tissues with active colitis produced large quantities of an inflammatory signaling molecule named interluekin-17 (IL-17), but very little IL-22, the latter of which has been linked to wound healing and mucus production. When worms recolonized the colon, however, immune cells began manufacturing much more IL-22. Blood profiling and genetic analysis further revealed that tissues in which helminths thrived increased carbohydrate metabolism—a prerequisite for mucus production. 

"Ulcerative colitis is often associated with decreased mucus production and the worms seem to somehow restore mucus production, possibly by inducing a population of immune cells that make IL-22," Loke says. "It's possible the mucus serves as a defensive barrier between bacteria and the gut that prevents bacteria from causing inflammation and crossing over into other tissues." Autoimmune diseases generally occur when the immune system overreacts to benign—and even beneficial—organisms living within the body. In the case of colitis, researchers suspect the reaction is directed toward the bacteria in the gut. Loke thinks that the human body may boost mucus production when it detects helminths as a defense against the parasites; for a patient with ulcerative colitis, the extra mucus may also help calm an excessively aggressive immune system.

"We saw an association with remission and immune cells that make IL-22, but we don't know for sure if these immune cells are actually induced by worms," Loke says. "You can't tell with a sample size of one," which is especially susceptible to the placebo effect. Still, Loke adds, "the results seems quite compelling, especially when you consider the background—all the animal studies and clinical trials that show worms can suppress colitis and other autoimmune disorders."

Mounting Evidence

In fact, in numerous animal studies, helminth infestation has protected rodents against colitis, asthma, rheumatoid arthritis, food allergies and type 1 diabetes.

Researchers have conducted few human studies, but most have shown promise. In aclinical trial published in 2005 in the journal Gut, Weinstock asked 29 participants with Crohn's disease (another autoimmune inflammatory bowel condition) to ingest 2,500 pig whipworm eggs every three weeks for six months. Twenty-three patients (79.3 percent) improved significantly, and 21 (72.4 percent) experienced remission. Both the researchers and participants, however, knew exactly what treatment they were receiving, which makes excluding a placebo effect impossible. 

In a controlled clinical trial published in 2005 in Gastroenterology, Weinstock and his colleagues gave 52 participants with colitis 2,500 pig whipworm eggs or a placebo every two weeks for three months. Thirteen of the 29 patients (44.8 percent) who received whipworm eggs improved, compared with only four of the 23 participants (17.4 percent) who received the placebo.

Weinstock and his collaborators point to these trials as experimental evidence that fits a global pattern: immune disorders are much rarer in less developed countries where helminthic infestation is widespread than in industrialized countries where much smaller populations host helminths. The "old friends hypothesis" proposes that the human immune system cannot learn to regulate itself without exposure to common pathogens like helminths that have coevolved with people and that modern hygienic practices deprive people of this necessary exposure, possibly explaining the relatively higher and more recent prevalence of immune diseases in industrialized countries like the U.S. 

Loke plans to continue researching helminthic therapy in people and in monkeys. "We are talking about doing a small trial of, say, 10 people and basically doing colonoscopies on them before and after giving them pig whipworm," he says. Loke also mentions that colitis plagues many juvenile monkeys in primate research centers and that he has received a pilot grant to treat diseased monkeys with human whipworm, an as-yet-unpublished experiment that is already returning promising results.

"When I first sat down to lunch with the guy who called me and he started telling me his story, I was really quite skeptical," Loke recalls. "But now I am completely changing my mind about helminthic therapy."

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http://www.functionalmedicine.org/ifm_ecommerce/Symp18Home.aspx

 

The Challenge of Emerging Infections in the 21st Century:
Terrain, Tolerance, and Susceptibility

This Symposium is about cracking the code of how susceptibility to infection or dysfunction is created. We will explore how a “hospitable host” is established through continuous and complex interplay between microbe exposure and an individual’s environment, lifestyle, and genetic profile.




What do we mean by Terrain, Tolerance, and Susceptibility?

Terrain: Terrain is the body ecology, the sum total of our 10 trillion cells and the 100 trillion microbes that live within and on us, and the environmental milieu in which all of these coexist.

Tolerance: The primary job of the immune system is not identification of that which is foreign; it is tolerance of that which is not foreign: our cells and commensal microbiota. Tolerance is controlled chaos within a fluid, ever-changing system.

Susceptibility: Susceptibility to disease and dysfunction is caused by our malleable genetic propensity, which is determined in large measure by what we eat, how we sleep, whom we associate with, and in general, how we live.

 

 

Programme: 

 

http://www.functionalmedicine.org/content_management/files/Symposium2011/IFM_18SymposiumBro_v5schedule.pdf

 

Intervenants : 

 

http://www.functionalmedicine.org/ifm_ecommerce/Symp18Speakers.aspx

 


 

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19 février 2011 6 19 /02 /février /2011 14:18

 

 

http://www.lymeinducedautism.com/images/LIATesting_handout_FINAL.pdf

 

 

 

Testing for Lyme Disease, Borreliosis and multiple-infections can be difficult to understand.

 

We hope that this guide will serve a purpose in narrowing down the lab testing and options available.

 

 

Lyme Disease / Borrelia Burgdorferi, Babesia, Anaplasma, Ehrlichia

and Bartonella

 

 

A Western Blot can be helpful for diagnosis. A positive diagnosis can be strongly considered by having at least one Borrelia specific band and symptoms. The Borrelia specific bands are: 18, 23/25, 30, 31, 34, 37, 39, 83 and 93.

If a band 30 or 31 shows up positive, then a viral panel should be done in order to rule out viruses such as HHV6, Cytomegalovirus, Epstein Barr, etc.

 A result such as this does not mean that the patient does not also have Borrelia.


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Voir http://www.lassesen.com/cfids/documents/Jadin_paper2.pdf

 

 

Summary: The possibility of a Rickettsial origin for symptoms of depression and psychotic disfunction has been suggested by French scientists (Ch. Nicolle, Giroud, Legag, Jadin, Bottero) in their published works. Hence 300 patients, diagnosed as suffering from depression, or other neuropsychiatric dysfunction have been treated with antibiotic where a positive indicaton of Rickettsial infection was revealed as follows:

1.Many symptoms of these patients were similar to those exhibited in chronic Rickettsial diseases. 2.The treatment followed the finding that their serum reacted positively to the Giroud micro-agglutination test.

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Grosse fatigue après une maladie infectieuse

Publié le 07/09/2006


Le syndrome de fatigue chronique (SFC) se définit par l’existence d’une asthénie persistante ou récidivante pendant au moins 6 mois, qui ne peut s’expliquer ni par une affection médicale ni par des troubles psychiatriques.

Le repos ou la diminution de l’activité ne permettent en aucun cas de venir à bout de ce syndrome proprement épuisant, autant pour le patient que pour le médecin.

 

Certes, le SFC peut survenir dans les suites d’une maladie infectieuse aiguë, mais de nombreuses études cas-témoins n’ont révélé aucune association significative entre ces deux cadres pathologiques.

 

Sur un autre plan, les états d’asthénie post-infectieuse sont un grand classique de la médecine et le spectre des infections causales est extrêmement large, puisqu’il inclut, par exemple, la brucellose, la maladie de Lyme ou encore la fièvre Q entre autres rickettsioses et la méningite virale, cette liste étant loin d’être exhaustive.

 

 

Quelle est la place des autres microorganismes, notamment des virus et quels sont les facteurs de risque associés au SFC ? Quelles sont son évolution à long terme et son expression symptomatique ? C’est à ces questions que répond une étude de cohorte prospective dans laquelle ont été inclus 253 malades résidant dans une région rurale d’Australie, en l’occurrence Dubbo. Dans tous les cas, ces sujets avaient été victimes d’une infection par le virus d’Epstein Barr, d’une fièvre Q ou encore d’une polyarthrite épidémique virale.

Au terme d’un suivi de 6 mois, il existait encore, chez 12 % des participants, des signes témoignant d’une maladie prolongée, caractérisée par une asthénie invalidante, des douleurs musculosquelettiques et des troubles neurocognitifs. Dans la majorité des cas (11 %), étaient réunis les critères diagnostiques requis pour évoquer le SFC.

Le phénotype de la fatigue post-infectieuse observée dans ce contexte s’est avéré stéréotypé et sa fréquence similaire, quelle que soit l’infection en cause. Plus que les caractéristiques démographiques ou psychologiques, ou encore les facteurs microbiologiques, c’est la sévérité de la maladie infectieuse inaugurale qui s’est révélée être la variable la plus prédictive d’un état d’asthénie prolongée.

Cette étude de cohorte prospective tend à faire de la fatigue post-infectieuse une entité pathologique à part entière qui n’est pas sans rappeler le SFC. Elle survient chez une minorité de malades victimes d’une infection aiguë virale ou non virale en règle sévère et peut perdurer au moins 6 mois. La physiopathologie  de ce curieux syndrome reste à déterminer.

 

Dr Peter Stratford

 

Hickie I et coll. : “Post-infective and chronic fatigue syndrome precipitated by viral and non-viral pathogens: prospective cohort study.” Br Med J 2006; publication avancée en ligne le 1er septembre 1/09/2006.

 

BMJ.2006 Sep 16;333(7568):575. Epub 2006 Sep 1.

Post-infective and chronic fatigue syndromes precipitated by viral and non-viral pathogens: prospective cohort study.

Hickie I,Davenport T,Wakefield D,Vollmer-Conna U,Cameron B,Vernon SD,Reeves WC,Lloyd A;Dubbo Infection Outcomes Study Group.

Brain and Mind Research Institute, Sydney University, Sydney, NSW 2050, Australia.

Abstract

OBJECTIVE: To delineate the risk factors, symptom patterns, and longitudinal course of prolonged illnesses after a variety of acute infections.

DESIGN: Prospective cohort study following patients from the time of acute infection with Epstein-Barr virus (glandular fever), Coxiella burnetii (Q fever), or Ross River virus (epidemic polyarthritis).

SETTING: The region surrounding the township of Dubbo in rural Australia, encompassing a 200 km geographical radius and 104,400 residents.

PARTICIPANTS: 253 patients enrolled and followed at regular intervals over 12 months by self report, structured interview, and clinical assessment.

OUTCOME MEASURES: Detailed medical, psychiatric, and laboratory evaluations at six months to apply diagnostic criteria for chronic fatigue syndrome. Premorbid and intercurrent illness characteristics recorded to define risk factors for chronic fatigue syndrome. Self reported illness phenotypes compared between infective groups.

RESULTS: Prolonged illness characterised by disabling fatigue, musculoskeletal pain, neurocognitive difficulties, and mood disturbance was evident in 29 (12%) of 253 participants at six months, of whom 28 (11%) met the diagnostic criteria for chronic fatigue syndrome. This post-infective fatigue syndrome phenotype was stereotyped and occurred at a similar incidence after each infection. The syndrome was predicted largely by the severity of the acute illness rather than by demographic, psychological, or microbiological factors.

CONCLUSIONS: A relatively uniform post-infective fatigue syndrome persists in a significant minority of patients for six months or more after clinical infection with several different viral and non-viral micro-organisms. Post-infective fatigue syndrome is a valid illness model for investigating one pathophysiological pathway to chronic fatigue syndrome.

PMID: 16950834 [PubMed - indexed for MEDLINE]PMCID: PMC1569956Free PMC Article

 

 

 

 

 

 

 

 


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