[New eating habits, new parasitic risks: The example of fish]. / De nouvelles habitudes alimentaires, de nouveaux risques parasitaires : l'exemple du poisson.

INTRODUCTION: In the past decades, the massive explosion of "Japanese" restaurants serving raw fish popularised new culinary habits in France. At the same time, consumers have made a habit of preparing raw or pickled fish dishes themselves at home. As a result, the identification of live parasitic worm larvae in raw fish flesh is common and a source of concern for professionals or amateur cooks. Sometimes, these worms are spit out or removed after fibroscopy in patients developing severe epigastric pain quickly after eating raw fish. This paper is aiming at having a quick review of the main parasites transmitted to humans by eating raw fish in France. METHODS: This article is based on the personal experience of the authors, on references preferentially from the French literature and on the results of the Fish Parasites (ANR) research program. RESULTS: From 2011 to 2014, Fish-Parasites (ANR) assessed the prevalence of parasitism in sea and freshwater fish belonging to 29 species. About 57% of sea fish were parasitised by Anisakidae. Larvae of Dibothriocephalus latus were found in pike, perch, and burbot in Lake Geneva but in none of the fish examined from Annecy or Le Bourget lakes. Concerning human anisakidosis, a retrospective survey was carried out in the years 2010 to 2014 among all medical parasitology laboratories from university hospitals in France. Thirty-seven cases of anisakidosis have been reported, including 18 cases of allergic anisakidosis. Six additional cases of severe Anisakidae allergy were reported to the National Allergovigilance Network over the same period. CONCLUSIONS: Despite the increase in consumption of raw fish, and compared to previous studies, cases of anisakidosis are decreasing, but their allergenic potential is increasing. The incidence of dibothriocephalosis, after some trend of emergence on the shores of Lake Geneva some 20 years ago, is currently decreasing, but sporadic cases of importation are still reported. Actions with professionals (investigation, providing of information) and research programs on management of parasitic risk control are being pursued and have resulted in an update of the technical instruction of the French General Directorate for Food on the control of parasitic risk in fish.

Culicoides and the Tartar Steppe: Il Deserto dei Tartari Culicoides and the spread of blue tongue virus.

Culicoides were described for the first time in England in 1713, but named by Latreille in 1809 only. Even so, they were better known as Ceratopogon until Kieffer reintroduced the name Culicoides. The family name became Ceratopogonidae, the description by Meigen (1803) being better adapted to that systematic level. Culicoides were considered simply as biting insects until it was found that they can carry filaria and viruses. In 1944, du Toit in Transvaal described their role in the transmission of blue-tongue virus. Blue-tongue disease has since extended progressively northward from South Africa, disseminated by Culicoides imicola. At the end of the 20th century, it reached the southern shores of the Mediterranean sea, and has since threatened the southern Europe. Surveillance and prevention procedures were put in place, but fortress Europe was taken breached when a different strain of the virus entered through Belgium in 2006. Transmitted by local Culicoides species that were aggressive and abundant, the disease spread quickly, in a disastrous epizootic southward through more than half of France. Westward, infected insects have been carried by wind over the Channel, introducing the disease to England.

Immunodiagnosis of Echinococcus infections: confirmatory testing and species differentiation by a new commercial Western Blot.

The Echinococcus Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from Echinococcus multilocularis, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to Echinococcus granulosus, and alveolar echinococcosis, due to E. multilocularis. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of Echinococcus-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between E. granulosus- and E. multilocularis-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available Echinococcus Western Blot IgG for the serological confirmation of human echinococcosis.

[Alveolar echinococcosis: a dreadful orphan disease]. / L'échinococcose alvéolaire. Une redoutable orpheline.

A WIDESPREAD DISEASE: Significant progress in screening for alveolar echinococcosis has reduced the number of new cases observed in Europe. Health education and serodetection campaigns have allowed earlier diagnosis and more effective treatment. The disease cannot however be totally eradicated due to the widespread wild reservoirs, sometimes even in the center of large cities. THERAPEUTICS: Early diagnosed and treatment can inhibit the inevitable progression observed after clinical manifestations appear. Drugs can block disease progression and surgical excision can be most effective. Inversely, the hopes raised by liver transplantation in patients with advanced stage disease have not been fulfilled due to the more or less late-onset metastasis favored by immunosuppressive treatments. PERSPECTIVES: There has been considerable progress in our knowledge of this parasite disease, particularly in improved diagnostic techniques. They have also demonstrated that humans are poor hosts for the parasite which is often spontaneously ejected. We are beginning to better understand the mechanisms of this spontaneous cure. Practical consequences would be a definition of receptive patient profiles or "vaccine" or immunotherapeutic procedures.

Echinococcus multilocularis infection in mice: in vivo treatment with a low dose of IFN-gamma decreases metacestode growth and liver fibrogenesis.

As no antiparasitic drug is definitively efficient in patients with alveolar echinococcosis, the effects of exogenous IFN-gamma on murine Echinococcus multilocularis infection were assessed with regards to the parasite burden, parasite-specific immune responses, and the urinary level of the collagen cross-link pyridinolines. They were analyzed after 3-week treatments with 1 or 5 micrograms of IFN-gamma per day twice a week. The treatment with 1 microgram transiently reduced the liver metacestode load, and the metastase weight as far as 6 weeks after the end of treatment. It slightly increased Th 1-type T cell responses and reduced the excretion of pyridinolines. These results should encourage further study to assess whether the decrease in liver fibrosis leads to an improvement of the efficacy of albendazole therapy. In contrast, the treatment with 5 micrograms increased the liver metacestode load and was less efficient than that with 1 microgram in decreasing pyridinoline excretion. These results incitate to follow up carefully patients with alveolar echinococcosis who are treated with IFN-gamma.

[Tinea capitis in Creteil. Trends over ten years]. / Teignes du cuir chevelu à Créteil. Evolution sur 10 ans.

INTRODUCTION: We analyzed tinea capitis data in a Paris suburban area over a 11-year period from (1985-1995) to evaluate epidemiology trends. PATIENTS AND METHODS: The following data were collected for patients seen at the Créteil myco-dermatology clinic with cultures positive for tinea capitis: sex, age, ethnic origin, fungal culture. RESULTS: Tinea capitis was observed in 336 cases (56 p. 100 females). Eight percent of the patients were under the age of 10 years and 11 p. 100 over 20 years. Trichophyton soudanense was isolated in 45 p. 100 of the patients. Anthropophilic agents rose over the 10 year period while the number of zoophilic agents remained stable. Specific dermatophytes appeared to predominate in populations of different ethnic origin. There was a two-fold increase in the number of tinea capitis cases in the 1990-1995 period compared with the five previous years. DISCUSSION: The percentage of adults with tinea capitis (11 p. 100) is higher than the 5 p. 100 reported in the literature. The rise in the number of anthropophilic tinea capitis cases resulted from an increase in T. soudanense (originating in Africa), probably related to the increasing immigrant population. This agent was identified in 95 p. 100 of the patients of African origin. Differing lifestyles and transmission between school children makes it quite difficult to interpret the correlation between ethnic origin and specific dermatophytes.

Leishmania infantum: lack of parasite resistance to amphotericin B in a clinically resistant visceral leishmaniasis.

Amphotericin B (AmB) has been used as a second-line treatment of visceral leishmaniasis, particularly in human immunodeficiency virus-positive patients. AmB median effective doses (ED50s) were determined on an isolate obtained before any treatment and on a second isolate obtained 4 years later from the same AmB-treated patient. ED50s were similar (0.059 and 0.067 mg/kg of body weight, respectively), demonstrating the first evidence of AmB ED50 stability of Leishmania infantum after a long-term drug exposure. An isoenzymatic study was performed in order to verify that the second isolate originated from the same parasite as the first isolate. The present case report showed that treatment failure was not due to parasite resistance in spite of a prolonged exposure to the drug.

Activity of a new liposomal formulation of amphotericin B against two strains of Leishmania infantum in a murine model.

The efficacy of a new liposomal formulation of amphotericin B was compared to that of amphotericin B deoxycholate (Fungizone) in a murine model of visceral leishmaniasis induced by Leishmania infantum. Median effective doses (ED50) were determined with two different strains: strain 1 was obtained from an untreated patient, and strain 2 was obtained from a patient who had received 12.5 g of amphotericin B over 3 years. BALB/c mice were infected intravenously on day 0 with promastigotes and then treated on days 14, 16, and 18 (strain 1) or on days 21, 23, and 25 (strain 2) with the liposomal formulation of amphotericin B (five doses were tested for each strain: 0.05, 0.1, 0.5, 0.8, and 3 mg/kg of body weight) or with conventional amphotericin B (four doses were tested for each strain: 0.05, 0.1, 0.5, and 0.8 mg/kg). Mice in the control group received normal saline solution. The liposomal amphotericin B formulation was about three times more active than the conventional drug against both strains. ED50 of the liposomal formulation were 0.054 (strain 1) and 0.194 (strain 2) mg/kg. ED50 of conventional amphotericin B were 0.171 (strain 1) and 0.406 (strain 2) mg/kg. Determination of drug tissular levels, 3 days after the last drug administration, showed a drug accumulation in hepatic and splenic tissues much higher after administration of liposomal amphotericin B than after conventional amphotericin B. A lack of toxicity was noted in all groups treated with the liposomal formulation.

Ultrastructural changes in parasites induced by nanoparticle-bound pentamidine in a Leishmania major/mouse model.

Drug targeting enhances drug efficacy. This principle was tested in the treatment of an experimental visceral leishmaniasis. Using transmission electron microscopy (TEM) we localized pentamidine-loaded polymethocrylate nanoparticles in the liver of mice infected with Leishmania major and compared the ultrastructural changes in the parasites of these mice when they were treated with bound versus free pentamidine. Between days 13 and 17 after infection, loaded nanoparticles treated group were injected i.v. with 3 doses of 0.17 mg/kg bound pentamidine loaded on 2 x 10(11) nanospheres; control groups received 2 x 10(11) unloaded nanospheres. Drug reference control groups received five doses of 200 mg/kg pentavalent antimony (Glucantime) or three doses of free pentamidine (0.17 mg/kg or 2.28 mg/kg). Mice treated with bound pentamidine displayed a 77% reduction in their parasite burden versus the untreated controls. Nanoparticles were located by TEM inside parasitized Küpffer cells, in the phagolysosomes without entering the Leishmania. The low dose of 0.17 mg/kg bound pentamidine damaged the Leishmania to the same extent as 2.28 mg/kg of free pentamidine (the usual dose in human chemotherapy). In the parasites inside the Küpffer cells, TEM showed a swollen mitochondrian with loss of cristae, destruction or fragmentation of the kinetoplast, loss of ribosomes and destruction of parasite structures except for the subpellicular microtubules. This study therefore shows that a dose of bound pentamidine 13 times smaller than the usual dose of free pentamidine has a similar effect on the parasite.

Tinea capitis in adults: misdiagnosis or reappearance?

BACKGROUND: According to the literature, tinea capitis in adults is supposed to be rare; we have recently observed a significant increase in cases. METHODS: Epidemiological, clinical and mycological features were studied in all adult tinea capitis diagnosed over 1 year in our department. RESULTS: Eight cases were observed: 75% of them were women, 50% never traveled and 62.5% had an underlying immunosuppressive disease. Scalp scaling and alopecia were the most frequent clinical features. A zoophilic dermatophyte was recovered in 50% of cases. CONCLUSION: These cases represent 11% of all tinea capitis observed in the same period of time (higher than the 3-5% observed in the literature). Secretion of sebum and colonization by Pityrosporon orbiculare are supported to protect the scalp against dermatophytic invasion after puberty, but an immune defect may also facilitate hair invasion. The erroneous notion of the disease being uncommon and the frequent atypical clinical presentation require a high degree of clinical suspicion.

Activity of pentamidine-loaded methacrylate nanoparticles against Leishmania infantum in a mouse model.

The use of drug delivery systems may reduce the toxicity and improve the activity of antileishmanial compounds. In view of such a strategy, we loaded the antileishmanial agent pentamidine on polymethacrylate nanoparticles. The activity of pentamidine-loaded nanoparticles was compared with that of free pentamidine in a BALB/c mice model of visceral leishmaniasis induced by Leishmania infantum. On day 0, mice were infected intravenously with 10(7) promastigotes and then treated via the tail vein on days 14, 16 and 18 with bound pentamidine, free drug or isotonic saline (control group). On day 21, liver parasite burdens were evaluated using the Stauber method. Livers and spleens were removed and weighed. Effective doses (ED) were determined using the Michaelis-Menten representation relating the percentage of parasite suppression to the dose. The ED50 of bound pentamidine was six times lower than that of free pentamidine (0.17 mg kg-1 vs 1.06 mg kg-1). The ED90 value calculated for bound pentamidine was 1 mg kg-1. It was not possible to obtain the ED90 for free pentamidine because the dose-response curve reached a plateau near 60% of parasite suppression. A significant decrease in liver and spleen weights, probably reflecting the leishmanicidal activity, was observed for treated mice with bound pentamidine. These results showed that bound pentamidine was more potent than the free drug against L. infantum in our BALB/c mice model.

Lack of H-2 gene influence on mouse susceptibility to secondary alveolar echinococcosis.

To determine whether the development of hepatic Echinococcus multilocularis infection is influenced by major histocompatibility-linked genes, metacestode growth and host immune responses were compared in 4 C57BL/10 congenic murine strains of H-2b, H-2d, H-2k and H-2q haplotypes. Although the H-2q strain appeared slightly more resistant than the other strains, the 4 strains of mice developed comparable spleen cell proliferative response and Th1/Th2 cytokine production at 13 weeks p.i. A kinetic analysis, performed in 2 of these congenic strains, showed a similar pattern of parasite growth in these mice and failed to detect any significant difference in the production of parasite-specific IgM, IgG1 and IgG2, antibodies. Consequently, this study indicates that the control of secondary alveolar echinococcosis is not H-2 gene-linked.

Activity of pentamidine-loaded poly (D,L-lactide) nanoparticles against Leishmania infantum in a murine model.

The activity of pentamidine-loaded poly(D,L-lactide) nanoparticles was compared, by determination of median effective doses (ED50), to that of free pentamidine in a murine model of visceral leishmaniasis induced by Leishmania infantum. BALB/c mice were infected intravenously on day O with promastigotes and then treated on days 14, 16, and 18. Groups of 5 mice received either 0.57, 1.14 and 2.28 mg/kg of free pentamidine (expressed in pentamidine base) or 0.055, 0.11, 0.22 and 0.44 mg/kg of pentamidine-loaded nanoparticles. In the control group, 12 mice received normal saline. The liver parasite burden was evaluated using the Stauber method 72 h after the last injection and drug levels in livers and spleens were determined. Bound pentamidine was 3.3 times more active than free drug (ED50 value = 0.32 mg/kg versus 1.05 mg/kg for free drug). Drug levels showed a weak accumulation in hepatic and splenic tissues following bound pentamidine administration. A lack of acute toxicity was noted in all groups treated by bound pentamidine. Results obtained with this biodegradable carrier may be of particular interest as no new major antileishmanial compound is today available.

Characterization of T-cell immune responses of Echinococcus multilocularis-infected C57BL/6J mice.

Specific and non-specific parasite-induced changes in lymphocyte responses were analysed in C57/BL/6J mice after intrahepatic infection with Echinococcus multilocularis. Spleen cells harvested at selected times after infection were in vitro stimulated with mitogens or a crude soluble parasite extract (EmAg) at an optimized dose. Cell proliferative responses to Con-A were not modified by the infection over the first 22 weeks. In contrast, LPS-induced responses were decreased from the 13th week. A strong CD4+ proliferative T-cell response to the parasitic extract of infected mouse spleen cells was observed at the early stage of infection. This response then progressively decreased but remained significantly higher than that of control mice until the 19th week of infection. Cytokine production was investigated after in vitro EmAg stimulation of spleen cells. IFN-gamma, IL-2, IL-5 were produced within the first weeks after infection whereas the detection of IL-10 was slightly delayed. Thus, the promotion of the disease does not appear associated with the expansion of one rather than another T-cell subset in C57BL/6J mice. A general immunosuppression affecting both mitogenic and parasite-specific T-cell responses was observed at the end of the infection.

PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients.

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.

Leishmaniasis in AIDS patients: results of leukocytoconcentration, a fast biological method of diagnosis.

Leukocytoconcentration is an easy, fast and inexpensive technique for the diagnosis of leishmaniasis from peripheral blood. The technique involves concentration of blood parasites on a small surface of a microscope slide while the red blood cells are removed by lysis. The results, compared with those of other methods (examination of cultures of blood samples and bone marrow smears), were very good and accurate. All but one of our cases of leishmaniasis were patients with HIV co-infection. Leukocytoconcentration facilitates follow-up of cases and fast detection of any relapse.

PIP: The biological diagnosis of an infectious disease is ideally based upon isolating and identifying the pathogenic agent in the host tissue and establishing cultures after direct examination under a microscope. That procedure allows both an accurate diagnosis and an epidemiological survey of the disease. When leishmaniasis occurs in an AIDS patient or any other immunocompromised patient, however, the procedure is often unsatisfactory for the following reasons: the samples are difficult to collect, there may be few parasites, and their growth is slow or impeded by other pathogenic agents. The clinical features, when they are not specific, may be attributed to an etiology other than leishmaniasis. This paper describes an easy, fast, and inexpensive technique for diagnosing leishmaniasis from peripheral blood. Leukocytoconcentration involves concentrating blood parasites on a small surface of a microscope slide while the red blood cells are removed by lysis. The results, compared with those derived from examining cultures of blood samples and bone marrow smears, were very good and accurate. All but one of the cases of leishmaniasis studied were patients co-infected with HIV. Leukocytoconcentration facilitates the follow-up of cases and fast detection of any relapse.