Confirmation and follow-up of neurocysticercosis by real-time PCR in cerebrospinal fluid samples of patients living in France.

Neurocysticercosis diagnosis is based on a combination of clinical, epidemiological, radiological, and immunological findings. We describe a real-time PCR assay for the confirmation of neurocysticercosis diagnosis in cerebrospinal fluid. The assay, tested on samples from nine patients living in France and diagnosed with neurocysticercosis, had a detection rate of 83.3% and 100% specificity.

Epidemiology and diagnosis of invasive pulmonary aspergillosis in bone marrow transplant patients: results of a 5 year retrospective study.

Of 322 patients undergoing allogeneic BMT, 18 developed invasive pulmonary aspergillosis at a mean of 115 days post-transplant, with a mortality rate of 82%. Pulmonary localization was common but cerebral involvement was seen in 10 of 18 patients. The diagnosis was made ante mortem in 11 patients by direct examination of pathological samples or culture and A. fumigatus was the only species isolated. Specific antibodies were not demonstrated before or at the time of clinical symptoms and Aspergillus antigen was only seen in one patient a few days before death.

Immunological study of hydatidosis. I. Evaluation of immunoelectrodiffusion tests and enzyme-linked immunoelectrodiffusion assay (ELIEDA) in human hydatidosis.

The comparative sensitivity, specificity and rapidity of immunoelectrodiffusion (IED) on cellulose acetate membranes and of immunoelectrophoresis (IEP) on agarose gel, were evaluated in the diagnosis of hydatidosis. Pooled crude fluid from several cattle hydatis cysts was used as antigen in tests on 1,750 non-hydatis and 400 hydatis sera obtained from patients with hepatic, pulmonary, splenic, peritoneal and cerebral hydatidosis before and after surgery. Coupled to a specific human reference serum for arc 5, IED shows a specificity comparable to that of IEP but its sensitivity is slightly higher and the amount of antigen needed is very small. The appearance of a typical "gloved finger" pattern in sera from patients with ruptured cysts emphasizes the interest of quick results (3 hours) obtained by this method. In order to increase the sensitivity of IED and to define the class of immunoglobulins involved in the antigen-antibody reaction, we have coupled this method to an enzymatic technique. The immune complexes precipitated by IED were treated with peroxidase-labelled antibodies specific to each class of human immunoglobulins. The specificity of this enzyme-linked immunoelectrodiffusion assay (ELIEDA) permits one to follow the immunologic evolution of hydatidosis and to identify IgM in ruptured hepatic cysts and IgA in pulmonary cysts.

Dirofilariasis of the breast in france.

Sections of a nongravid adult female Dirofilaria, probably D. repens, were found within a subcutaneous nodule excised from the breast of a 45-year-old Frenchwoman. This is the 29th case of D. repens in man in France and the first in which the parasite was located in the breast. The patient apparently acquired the infection while vacationing in the southeastern Mediterranean region of the country.

Experimental pathogenicity of viscerotropic and dermotropic isolates of Leishmania infantum from immunocompromised and immunocompetent patients in a murine model.

The pathogenicity of 22 strains of Leishmania infantum from 11 HIV-infected and 11 immunocompetent patients with visceral (VL, n = 16) or cutaneous (CL, n = 6) leishmaniasis, belonging to 3 zymodemes (MON-1, n = 14; MON-29, n = 5; MON-33, n = 3), was studied using a murine model. For each strain 16-20 BALB/c mice were infected at day 0 (d0) by i.v. injection of 10(7) stationary-phase promastigotes. Parasite burdens were quantified in the spleen and liver of 4-5 mice of each strain at d7, d20, d60 and d90 or d100, using a sensitive culture microtitration technique. A great variability of infection profiles between strains was observed: (i) six strains showed a progressive infection, with a predominance of hepatic parasites at d7 or d20 (10(4)-10(6) g-1), then a continuous rise of splenic parasites reaching 10(5)-10(7) g-1 at d90 or d100 contrasting with a stagnation or decrease in the liver; (ii) ten strains gave a controlled infection with hepatic parasite burden reaching 10(4)-10(5) g-1 at d7 or d20, followed by a more or less rapid decline leading frequently to no detectable parasites; (iii) six strains resulted in other profiles, i.e., undetectable infection (n = 1) or low parasite loads (n = 4), or late occurrence of parasites in the spleen (n = 1). No relationship was observed between profile and growth characteristics in vitro or zymodeme of the strain. Strains originating from CL never gave a visceralizing pattern in mice, but belonged more frequently to the avirulent type compared to VL strains. Strains from HIV-infected patients were not less virulent than those from immunocompetent individuals. These results showed that the course of L. infantum infection varies markedly with intrinsic parasite factors that display striking intraspecific variability.

Influence of the host and parasite strain in a mouse model of visceral Leishmania infantum infection.

We investigated the respective roles of the host and parasite strain in a murine model of visceral leishmaniasis. Balb/c and C57Bl/6 mice were selected for their respective 'non cure' and 'cure' haplotypes vis-a-vis Leishmania major. Mice were infected with 10(7) stationary-phase promastigotes of four strains of Leishmania infantum with different infection profiles in mice: visceralization or regulation, as established by Sulahian et al. (Sulahian et al. (1998) FEMS Immunol. Med. Microbiol. 17, 131-138). The infection was monitored by measuring parasite load in the liver and spleen on days 9, 22, 44 and 87 post-infection, using a sensitive microtitration technique. Similar profiles (visceralizing or regulating) were observed in the two mouse strains, suggesting a predominant role of the Leishmania strain in the visceralization process. The host response was assessed by analyzing the granulomatous response in the liver and by quantifying specific IgG, IgG1 and IgG2a as a marker of the Th1/Th2 immune response. A granulomatous response was observed in both strains of mice but was more pronounced with visceralizing strains of L. infantum and in C57Bl/6 mice compared to Balb/c mice. The kinetics of anti-Leishmania IgG antibody production was similar in all the groups, but the distribution of IgG1 and IgG2a isotypes was different between the two mouse strains: Balb/c mice had a predominantly Th2-like response whereas C57Bl/6 had a mixed Th1/Th2-like response. This study demonstrates the determining role of both the parasite and mouse strain in the outcome of L. infantum infection. The Th1/Th2 concept does not seem to explain susceptibility and resistance to infection in our model of visceral L. infantum infection, contrary to the L. major model.

The use of ELIEDA (enzyme-linked-immuno-electro-diffusion-assay)) in the study of humoral antibodies in human schistosomiasis.

In order to improve the sensitivity of immuno-electrodiffusion (IED) we have developed a technique of treating the precipitated immune complexes with enzyme-labelled antibodies. This technique we term ELIEDA (Enzyme-Linked-Immuno-Electro-Diffusion-Assay). The enzyme-labelled antibodies can be made monospecific for each class of immunoglobulins found in patients with schistosomiasis and which precipitate with schistosome antigens. We believe that this sensitive technique will prove valuable in studying the sequential development of antibodies in human and experimental schistosomiasis.

Lipid formulations of amphotericin b in the treatment of experimental visceral leishmaniasis due to Leishmania infantum.

Despite significant antileishmanial activity of amphotericin B (AmB) in vitro, the use of the deoxycholate formulation (Fungizone) is limited because of serious side effects. Lipid formulations of AmB have been proposed to reduce this toxicity. We compared the tolerance and efficacy of the conventional AmB prepared with deoxycholate, AmB emulsified in Intralipid 20%, amphotericin B lipid complex (Abelcet), and liposomal AmB (AmBisome) in a murine model of visceral leishmaniasis induced by Leishmania infantum. Control groups included untreated mice and mice treated with the pentavalent antimonial (Glucan-time). Balb/C mice were infected intravenously on day 0 with 10(7) promastigotes of L. infantum, then treated from days 7 to 17 (early treatment group) or from days 60 to 70 (delayed treatment group). Glucan-time was administered daily by intraperitoneal injection, whereas AmB formulations were administered intravenously on alternate days. On days 20, 60 and 120 in the early treatment group and 72 and 125 in the delayed treatment group, parasite burdens were determined in liver, spleen, and lungs by subculturing using a microtitration method. Abelcet (12 mg/kg) and AmBisome (12 mg/kg) completely eradicated the parasites from the tissues. Both of these lipid formulations enabled higher dosages to be tolerated, and were remarkably more effective than Fungizone (0.8 mg/kg) and AmB diluted in Intralipid 20% (1.2 mg/kg) in the treatment of murine visceral leishmaniasis due to L. infantum.

Effects of immunosuppressive therapy on murine Leishmania infantum visceral leishmaniosis.

We evaluated the effect of immunosuppressive therapy on the course of infection, the spleen cell immunophenotype and cytokine production during murine Leishmania infantum visceral leishmaniosis (VL). Rousseau et al. [1] recently reported that prolonged administration of dexamethasone induces limited reactivation of chronic murine visceral leishmaniosis, with no clear Th1-Th2 cytokine patterns. We found that another glucocorticoid, hydrocortisone acetate, had similar effects during acute visceral leishmaniosis, i.e. an increase in parasite burden in the spleen, but not the liver, of infected mice. A significant increase in parasite burden in both the liver and the spleen was only achieved when mice were treated with combined dexamethasone + pentoxifylline immunotherapy; increases in parasite burden were never associated with a specific spleen cell immunophenotype or a Th1-Th2 cytokine secretion profile.

Microplate method for obtaining Leishmania clonal populations.

The various clinical expressions observed in human leishmaniases result from complex host-parasite relationships in which the biodiversity of the parasite is a determining factor. Because Leishmania strains isolated from humans are composed of heterogeneous populations, it is crucial to use clonal lineages for studies on the characterization of these parasites. Presently, techniques used for cloning Leishmania spp. parasites are time-consuming and show poor efficiency. Here, a method developed in 96-well microplates is described, which allows one to rapidly obtain numerous clones of Leishmania in the most versatile and efficient way. The technique may be useful for cloning various protozoa as well as Leishmania spp.

[Techniques applicable to broncho-alveolar lavage fluid in hospital laboratory for the diagnosis of parasitic and fungal pneumopathies in immunosuppressed patients]. / Techniques applicables au liquide de lavage broncho-alvéolaire dans un laboratoire hospitalier pour le diagnostic des pneumopathies parasitaires et fongiques chez les malades immunodéprimés.

Parasitic and fungal organisms which are likely to cause pulmonary infections in immunosuppressed patients can be detected in broncho-alveolar fluid (BAL fluid). Single and standard methods, such as direct examination of the pellet, eosine-methylene blue fast (RAL 555), cultures in usual mediums of mycology must be systematically applied to this sample and may help detect these organisms without further exploration. If the results are negative, more recent techniques can be used if they present a real asset: an easier reading and mostly an improved sensitivity. Such is the case of immuno-fluorescence assay with monoclonal antibodies for detection of Pneumocystis carinii, and inoculation of MRC5 fibroblast cell line in tissue culture for isolation of Toxoplasma. Fungal pulmonary infections diagnosis has not yet succeeded in benefiting from modern findings: latex tests proposed for the detection of circulating antigens are nor sensitive nor specific, except the "Crypto LA test". Considering the relatively frequent association with other infectious agents, the detection of a parasitic or fungal organism in the BAL fluid should not interrupt investigation of this sample; neither should it lead to hasty conclusions regarding the responsibility of this agent in acute pneumopathy. This role will have to be evaluated according to criteria which are different for each isolated organism.

[Appendiceal localization of bilharziasis: value of extemporaneous histological examination]. / Localisation appendiculaire de la bilharziose: valeur de l'examen histologique extemporané.

The association of appendicitis with schistosomiasis in the appendix is extremely rare in France. We report herein a new case in which the diagnosis was made, in the presence of pseudo-tumoral appendicitis, by frozen section biopsies. The patient presented with a typical acute appendicitis, without urinary symptoms, and with granulocytosis (14000 white cells/mm3, without eosinophilia). At laparotomy, the appendix was voluminous, with necrotic abscess, and lymph node masses were noted on the greater omentum. Frozen section biopsies of an omental tumor showed schistosoma eggs, without malignant cells. Appendicectomy and partial omentectomy were performed. Postoperative course was uneventful. Pathologic examination of the resected specimen showed schistosoma eggs in all layers of the appendix, and in the omentum. Final diagnosis was established by positive serology and by findings Schistosoma haematobium eggs in the urine. The patient was treated postoperatively by praziquantel. In conclusion, in case of acute appendicitis, with pseudotumorous appendix and lymph node masses, even with poor epidemiological findings on the clinical history, frozen section biopsies can sometimes establish the diagnosis of schistosomiasis with appendicitis, and avoid unjustified bowel resection.

Therapeutic outcome and prognostic factors of invasive aspergillosis in an infectious disease department: a review of 34 cases.

BACKGROUND: Invasive aspergillosis (IA) is one of the most frequent, feared and life-threatening opportunistic infection in immunocompromised patients. We wished to assess the therapeutic outcome and identify prognostic factors of IA. METHODS: We reviewed retrospectively all patients managed in our department for a proven or probable IA over the last 10 years. RESULTS: A total of 34 patients were identified: 20 hematopoietic stem cell recipients, 7 infected with the human immunodeficiency virus, 6 hematological malignancies, and only 1 diabetes mellitus. IA involved the lower respiratory tract in all but one case with sinonasal infection. Among patients with pulmonary IA, sinuses were involved in four cases and the brain in five cases. First line antifungal therapy included amphotericin B deoxycholate (56%) or its lipid formulations (18%), itraconazole (15%) and voriconazole (12%). Eight patients also underwent surgery. Median survival was only 64 days and 73% of patients died during follow-up. A favorable outcome of IA was documented in only 48% of patients. Multivariate analysis identified neutropenia as the only factor associated with unsuccessful outcome (p = 0.003). CONCLUSIONS: IA remains therefore associated with a highmortality rate, especially in patients with neutropenia.

Late-onset eosinophilic chronic meningitis occurring 30 years after Taenia solium infestation in a white Caucasian woman.

Unlike solitary parenchymal cysts, chronic meningitis is unusual in patients with neurocysticercosis and may poorly respond to treatment. We report the case of neurocysticercosis characterized by severe headache and chronic eosinophilic meningitis occurring 30 years after infestation with Taenia solium. The patient showed considerable improvement following treatment with albendazole and prednisone.

Longitudinal study of the specific humoral and cellular response to Toxoplasma gondii in a patient with acquired toxoplasmosis.

Specific cellular and humoral response to Toxoplasma were evaluated during a 2 yr follow up in a patient with acquired infection. Antibodies were titrated using conventional serological tests and analyzed by western blot technique. Analysis of the patterns obtained with IgA, IgG and IgM antibodies allowed a clear differentiation between acute and chronic stages of infection. Determination of lymphocyte subsets showed an important increase in the number of CD8 suppressive cells at the acute phase whereas CD4 cells remained within the normal range. Lymphocyte proliferation to Toxoplasma antigen was detectable one month after the onset of clinical symptoms and remained constantly positive. These results indicate that western blot analysis of antigens recognized by antibodies and determination of lymphocyte subsets may be helpful in the characterization of the infectious stage of acquired toxoplasmosis.

Activity of IFN-gamma in experimental Leishmania infantum murine visceral leishmaniasis.

The effect of recombinant interferon-gamma (rIFN-gamma) on Leishmania infantum infection was investigated in vivo. BALB/c mice were injected intravenously (i.v.) with 10(7) promastigotes of Leishmania infantum. rIFN-gamma, 10(6) U given intraperitoneally (i.p.) daily on 3 consecutive days or 4 times on alternate days from day 7 (d7) post infection, had no detectable effect on the parasite burdens in liver, spleen, and lungs as compared to untreated mice. However rIFN-gamma enhanced the activity of Glucantime (50 mg/kg/d intraperitoneally for 7 days) in the liver and in the lungs. The additive effect of rIFN-gamma was still observed at day 30 post-infection, i.e. 15 days after cessation of therapy. By contrast the combination of the two drugs had no activity against splenic parasites.

[A critical study of immuno-enzymatic reactions coupled with the precipitation tests on cellulose acetate membranes]. / Etude critique des réactions immuno-enzymatiques couplées aux tests de précipitation sur membranes d'acétate de cellulose.

Precipitating tests carried out on cellulose acetate membrane can be increased by treating the immune complexes with enzyme linked anti-immunoglobulin antibodies. From our trials in parastic diseases (Amoebiasis, schistosomiasis, fasciolasis, filariasis, hydatidosis, trichinosis) and mycosis (Aspergillosis, Candidiasis), it seems that the immuno-enzymatic labelling of the "active" precipitating reactions should only be taken in consideration. We must insit on the importance of ELIEDA (enzyme-linked-immuno-electrodiffusion-assay) and ELIDEPA (enzyme-linked-immuno-double-electro-phoresis-assay). Both of these analytical assays are particularly sensitive. The sequence of appearance of the multiple precipitating systems of the complex parasitic mosaic can easily be watched. The different classes of immunoglobulins and their kinetics are determined by the use of monospecific antibodies linked to different enzymes which give a polychromic specific staining.

Efficacy of aminosidine administered alone or in combination with meglumine antimoniate for the treatment of experimental visceral leishmaniasis caused by Leishmania infantum.

BALB/c mice with an experimental visceral leishmaniasis produced by Leishmania infantum were treated with aminosidine sulphate alone or combined with meglumine antimoniate. Parasite burdens in the liver and spleen were determined by subculturings using a sensitive microtitration method. Treatments with aminosidine alone decreased the parasite burdens compared with those observed in the untreated mice, but were less efficacious than meglumine antimoniate. Aminosidine combined with meglumine antimoniate resulted in an increased efficacy compared with either drug given alone. However, these regimens were associated with toxicities and with persistence of hepatic and splenic leishmanial foci after drug administrations.