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.

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.

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.

Virulence of Leishmania infantum is expressed as a clonal and dominant phenotype in experimental infections.

Human Leishmania infantum infection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence in L. infantum visceral leishmaniasis is clonal and dominant in nature.

Real-time PCR as a new tool for quantifying Leishmania infantum in liver in infected mice.

The parasitic loads of mouse livers experimentally infected with Leishmania infantum were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The Leishmania DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.

Value of antigen detection using an enzyme immunoassay in the diagnosis and prediction of invasive aspergillosis in two adult and pediatric hematology units during a 4-year prospective study.

BACKGROUND: Invasive aspergillosis (IA) is a well recognized, life-threatening infection in neutropenic patients and stem cell transplantation recipients. Early diagnosis is important to achieve the best outcome for these patients; however, definite proof often is difficult to obtain due to counterindicated invasive procedures. METHODS: This study evaluated the specificity and sensitivity of the detection of galactomannan (GM) for the diagnostic and prediction of IA in 347 children from the Pediatric Hematology Service and 450 patients from the Bone Marrow Transplantation Unit at the Hôpital Saint-Louis in Paris. Serial screening of Aspergillus GM circulating antigen was evaluated using a double sandwich ELISA assay (Platelia Aspergillus) on 6209 sera. Among the patients studied, 53 presented with confirmed IA (n = 27 patients) or probable IA (n = 26 patients). RESULTS: Antigen was detected on at least two sequential sera in 48 of 53 patients, with a sensitivity of 90.6%. GM antigenemia was detected before the onset of radiologic signs in 31 of 48 patients (64.6%), with a mean of -8.4 days, and before clinical symptoms in 18 of 48 patients (39.6%), with a mean of -6.9 days. In patients without IA, 44 of 744 had positive antigenemia, resulting in a specificity of 94%. False positive results could not be related to the presence of a concurrent mucositis. CONCLUSIONS: This large, prospective study allowed the authors to define better the conditions for the use of GM immunocapture ELISA in surveying patients who are at high risk for IA. The presence of antigen has a good diagnostic value mainly when there is an increase in the titer on two consecutive sera samples. A repeated negative result is a strong argument against the diagnosis of IA; however, an awareness of the possibility of unexplained false negative results is important.

Assessment of Leishmania promastigote growth in vitro by means of nucleoside hydrolase activity determination.

Nucleoside hydrolases (NH) are involved in the purine salvage pathway of protozoan cells for the biosynthesis of nucleic acids. We developed a simple and reliable microassay based on N-ribohydrolase dosage using 4-nitrophenyl-beta-D-ribofuranoside (NPR) substrate for the quantification of Leishmania infantum. The free promastigote stage of L. infantum contains high amounts of NH capable of cleaving NPR, but the parasitic amastigote does not. The method allows reliable quantification of viable parasites over a wide range of concentrations (5 x 10(4) 2 x 10(8) parasites ml(-1)) in a single assay. No difference in NH activity was observed between various strains at equivalent concentrations and growth curves determined with NH dosage were similar to optical counts. Samples can be stored at -20 degrees C for weeks before use in this unique assay without significant loss of NH activity. The method is particularly simple and versatile and proves well adapted for the determination of growth characteristics and drug screening studies of L. infantum promastigotes in vitro.

Experimental pathogenicity of a presumed monoxenous trypanosomatid isolated from humans in a murine model.

Two strains of a presumed lower trypanosomatid isolated from immunocompetent and HIV-infected humans in French West Indies were investigated in vitro and in vivo in a murine experimental model. The ability of parasites to grow in vitro in bone marrow-derived macrophages and their virulence in vivo were assessed. For in vivo infection, two groups of BALB/c mice were inoculated either by the subcutaneous or intravenous route with 10(7) promastigotes at day 0. Infection was monitored by measuring parasite load in liver, spleen, foot pad, popliteal, and mesenteric lymph nodes and brain from day 7 to day 150 post-infection using a microtitration technique. Parasites multiplied in mouse macrophages in vitro. In vivo, both strains proved infective to mice and capable of visceralization and dissemination in the popliteal and mesenteric lymph nodes, liver, spleen, and even brain. Both strains elicited a strong humoral response against trypanosomatid antigen in mice, which cross-reacted with Leishmania antigen. Contrasting with the straightforward dissemination of parasites, the infection was strikingly well tolerated by the murine host with no clinical signs and minimal tissue changes around parasitized macrophage infiltrates.

Evidence for determining parasitic factors in addition to host genetics and immune status in the outcome of murine Leishmania infantum visceral leishmaniasis.

C.B-17 SCID and congenic BALB/C mice were used to examine Leishmania infantum strain pathogenicity independently of host genetic factors. While parasite loads were significantly higher in immunodeficient mice than in immunocompetent mice, the kinetics of infection during a long-term follow-up were similar, suggesting that intrinsic parasitic factors also influence the outcome of L. infantum infection.

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.

Experimental evaluation of second-line oral treatments of visceral leishmaniasis caused by Leishmania infantum.

In a murine model of Leishmania infantum visceral leishmaniasis, metronidazole, ketoconazole, fluconazole, itraconazole, and terbinafine were less effective than antimonial agents in reducing hepatic parasite load. Ketoconazole potentiated the effect of meglumine antimoniate reference therapy through its marked activity against spleen infection.

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.

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.

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.

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.

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.

Therapeutic effect of reference antileishmanial agents in murine visceral leishmaniasis due to Leishmania infantum.

A sensitive, culture-based, microtitration technique has recently been developed for determining parasite burdens in organs recovered from Balb/c mice infected with Leishmania infantum. In the present study, this technique was used to examine the efficacy of three, first-line, antileishmanial agents in reducing parasite burdens and eradicating parasites from target organs in mice. Treatment with meglumine antimoniate (50 mg SbV/kg.day) significantly reduced the parasite burdens in the livers and lungs (by about 10-fold and > 100-fold, respectively) but not those in the spleens. Although use of a higher dose of meglumine antimoniate (200 mg SbV/kg.day) resulted in an even more dramatic reduction in the parasite burdens in the livers, it had no significant effect on the burdens in the spleens. Treatment with amphotericin B (0.8 mg/kg every other day) resulted in significant reductions in the parasite burdens in the livers, spleens and lungs of infected mice. Although low doses of aminosidine (20 mg/kg.day) had no effect, high doses (200 mg/kg.day) resulted in undetectable parasite burdens in the livers, for at least 100 days post-treatment, and marked reductions in burdens in the spleens. These results are consistent with previous data from studies using animal models of visceral leishmaniasis. Thanks to the sensitivity of the technique, culture microtitration revealed that none of the drug schedules achieved the elimination of all parasites in all target organs. The murine model used mimics some important features of HIV/Leishmania infantum co-infections in humans.

Therapy of visceral leishmaniasis due to Leishmania infantum: experimental assessment of efficacy of AmBisome.

The tolerance and efficacy of amphotericin B (AmB) deoxycholate (Fungizone) were compared with those of liposomal AmB (AmBisome) in a murine model of visceral leishmaniasis induced by Leishmania infantum. Control groups consisted of untreated mice and mice treated with a pentavalent antimonial (Glucantime). BALB/c mice were infected intravenously on day 0 with 10(7) promastigotes of L. infantum and then treated from day 7 to 17 (early treatment group) or from day 60 to 70 (delayed treatment group). The pentavalent antimonial was administered daily by intraperitoneal injection, whereas AmB formulations were administered intravenously on alternate days. On days 20, 60, and 120 (early treatment group) and on days 72 and 125 (delayed treatment group), parasite burdens in the liver, spleen, and lungs were determined by subculturings using a microtitration method. A dose range study showed that administration of AmBisome at the well-tolerated doses of 5 or 50 mg/kg of body weight completely eradicated the parasites from the tissues. At 0.8 mg/kg, AmBisome proved more efficacious than AmB deoxycholate administered at the same dose. We also compared the levels of AmB deoxycholate and AmBisome in plasma and tissue. Mice treated with AmBisome had levels of AmB in tissue much higher than did AmB deoxycholate-treated mice with persistent detectable levels 14 weeks after treatment. These results seem to account for the remarkable efficacy of the liposomal formulation of AmB in the treatment of visceral leishmaniasis due to L. infantum.

Comparison of an enzyme immunoassay and latex agglutination test for detection of galactomannan in the diagnosis of invasive aspergillosis.

Aspergillus antigenemia was followed up in 215 consecutively observed bone marrow transplant (BMT) patients over a period of two years, using both a latex agglutination test and a sandwich immunocapture enzyme immunoassay (EIA) with a rat antigalactomannan monoclonal antibody as capture and detector antibody. For each patient, sequential sera (3 to 20) were obtained before and after BMT. No positivity was observed before BMT. After BMT, the EIA and latex agglutination test were positive in 19 and 4 patients respectively of 25 patients with confirmed aspergillosis and 14 and 7 of 15 patients with probable aspergillosis. In 19 of 25 patients with confirmed aspergillosis and 9 of 15 patients with probable aspergillosis, the EIA was more sensitive and detected infection earlier than the latex test. In all positive cases, antigenemia rapidly increased in sequential samples and remained strongly positive. In 31 of 169 (19%) BMT patients without clinical signs of aspergillosis, the EIA was occasionally positive in samples taken within the first month after BMT, giving a specificity of 81% in these patients. In non-BMT patients suffering from other diseases (n = 77), the specificity was 98.7%. The overall positive and negative predictive values for the EIA were 54% and 95% respectively. These results favour the use of EIA for early diagnosis and monitoring of aspergillosis in BMT patients, although the predictive value of transient positivity remains to be ascertained.