Assessment of cryptodiag for diagnosis of cryptosporidiosis and genotyping Cryptosporidium species.

The performance of a new commercial PCR-enzyme-linked immunosorbent assay (ELISA) (Cryptodiag; Bio Advance, France) for the diagnosis of cryptosporidiosis and the identification of Cryptosporidium hominis and C. parvum from stool samples was examined. This test is based on PCR amplification of Cryptosporidium DNA extracted from stools, followed by an ELISA based on hybridization with Cryptosporidium sp.-, C. hominis-, or C. parvum-specific probes. In spiking experiments, approximately five oocysts were detected either in water or in stool suspensions while assessing for the efficient removal of stool PCR inhibitors. No cross-reactivity was observed in the detection of C. parvum and C. hominis using the respective specific probes. Thirty-three fecal samples from patients with microscopically proven cryptosporidiosis and 118 from patients with or without other digestive protozoan infections were tested by Cryptodiag, blinded to the results of microscopy. Compared to microscopy, the sensitivity of Cryptodiag was 97.0% (32/33) and 100% (33/33), including the gray zone, and specificity was 98.3% (116/118) and 96.6% (114/118), including the gray zone. Among 34 positive results, Cryptodiag identified 19 due to C. hominis, 8 due to C. parvum, and 7 due to Cryptosporidium spp. Genotyping by Cryptodiag agreed with reference typing methods in 85% of cases of C. parvum or C. hominis infections. Cryptodiag proved to be reliable and sensitive for the diagnosis of cryptosporidiosis. The use of specific probes allowed the identification of C. hominis and C. parvum, i.e., the two main species responsible for human cryptosporidiosis, and rapidly provided information on the possible source of infection.

Cutaneous sparganosis in France: the second case described from Europe. Case report.

The first case of sparganosis is reported from France. The patient, a 21-year-old man, presented with a subcutaneous lump on the chest, and the diagnosis was made on histological examination after needle biopsy. He achieved a complete recovery.

Prophylaxis of toxoplasmosis infection with pyrimethamine/sulfadoxine (Fansidar) in bone marrow transplant recipients.

Prophylaxis against toxoplasmosis with weekly administration of pyrimethamine/sulfadoxine (Fansidar) was assessed for efficacy and toxicity in bone marrow transplant (BMT) recipients over a 21 month period. Sixty-nine of 90 consecutive seropositive patients were evaluable. Fansidar was administered from the time of established engraftment (median day 40, range days 13-100). Medication was scheduled to be continued until 6 months or longer in cases of continued immunosuppression (median 10 months, range day 72 to 22 months). No proven case of toxoplasmosis occurred in patients receiving prophylaxis. In addition, there were no cases of Pneumocystis carinii. Side-effects included BM suppression requiring cessation (n = 4) or interruption (n = 8) of therapy and rash (n = 1). To evaluate toxicity associated with prolonged therapy, 42 evaluable patients were assessed at 6 months following transplant (or at least 4 months of continuous treatment). Haematological toxicity was minimal and compounded in three patients showing moderate derangement by cytomegalovirus infection and graft-versus-host disease. Fansidar is an effective prophylactic agent against toxoplasmosis in BMT patients.

Detection by PCR of Toxoplasma gondii in blood in the diagnosis of cerebral toxoplasmosis in patients with AIDS.

The polymerase chain reaction (PCR) for amplification of Toxoplasma gondii DNA was performed prospectively in the blood of 19 patients with AIDS and cerebral toxoplasmosis. The B1 gene and TGR1E sequence were used as targets and results were confirmed by hybridisation. Controls consisted of 24 HIV infected patients with tissue culture proven T gondii parasitaemia and 57 HIV infected patients without toxoplasmosis. PCR was positive with both targets in 20 of 24 samples (84%) from patients with parasitaemia. Three of 57 samples (5%) from patients without toxoplasmosis were PCR positive with either target, but none was positive with both targets. Only three of the 19 patients (16%) with cerebral toxoplasmosis had a positive PCR with both targets before the start of specific treatment. PCR performed in blood is of little diagnostic value in cases of cerebral toxoplasmosis but could be useful in patients with disseminated 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.

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.

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.

Early kinetics of Toxoplasma gondii infection in mice infected orally with cysts of an avirulent strain.

We determined the early kinetics of Toxoplasma gondii infection in Swiss Webster mice inoculated with the avirulent C strain by counting parasites in the blood, spleen, Peyer's patches, liver, lungs, and brain. Animals were orally inoculated with cysts on day zero (D0), and parasites were counted using a tissue culture method at 12, 24, and 36 hr and 2, 3, 7, 10, 14, 21, 30, 50, and 72 days postinfection. The spleen and Peyer's patches were the first organs found parasitized, on D2 and D3, respectively, followed by the lungs and liver on D7 and the brain on D10. No parasitemia was detected. This suggests that early dissemination of this avirulent strain from the intestine into the general circulation occurs essentially via the lymphatic system. Parasites persisted at a high level in the brain during the chronic phase. In the lungs, parasites were no longer detected by D72, while parasite numbers initially declined in the spleen and Peyer's patches but then showed a second peak, possibly due to recirculation of T. gondii. These results suggest that lymphoid organs play a key role in T. gondii dissemination during the acute phase and may also constitute a persistent source of parasite resurgence.

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.

[Experimental models of toxoplasmosis. Pharmacological applications]. / Modèles expérimentaux de toxoplasmose. Applications pharmacologiques.

Toxoplasma gondii is an ubiquitous protozoan parasite causing severe or life-threatening infections in immunocompromised patients and in congenitally infected infants. Animal models have been extensively used to describe the pathology of infection and to identify new effective drugs for the treatment of congenital infections, chrorioretinitis and toxoplasmic encephalitis. Although inherent differences between man and animal can reduce the relevance of data obtained experimentally, animal models have greatly improved our knowledge on the various aspects of toxoplasmosis. Toxoplasma infection can be easily obtained in most laboratory animals, with exception of rats which are partially resistant. According to the strain used, the resulting infection may be acute, subacute or chronic, and can be monitored either by the survival of animals, the histopathological examination of lesions or, preferably, by titration of parasites in infected tissues using subinoculation to mice or tissue culture. This latter method has proved particularly useful to describe the kinetics of infection in host tissues and to assess the efficacy of drugs, according to their pharmacokinetics and tissue distribution. The relevance of results obtained in animal models of congenital toxoplasmosis and of chrorioretinitis is more questionable, due to the marked differences between the mode of infection in humans and in animals. Experiments performed in primates provided valuable informations for the management of therapy of congenital toxoplasmosis but were of limited interest for ocular toxoplasmosis. The pathogeny of toxoplasmic encephalitis is still poorly understood, and no experimental model is fully satisfactory to produce focal encephalitic lesions as observed in immunocompromised humans. Acute infections with highly virulent strains induce disseminated infection with major pulmonary and brain involvement, and thus can be used to assess the efficacity of drugs in these tissues. Direct inoculation of tachyzoites into brain tissue can induce focal encephalitis but this model is of difficult use for large scale studies. Although cellular immunity is mainly responsible for the control of toxoplasmosis at the chronic stage, administration of immunosuppressive drugs does not usually result in focal brain reactivation; such reactivation can only be obtained using antibodies against CD8 and CD4 T lymphocytes or interferon gamma. Another experimental approach is the use of genetically immunodeficient animals: these models are of limited interest for pharmacological research since infection of nude or T depleted mice usually results in a dissemination of infection; however, using these models it could be clearly demonstrated that immunity plays a major adjunctive role in the control of acute infection. Concurrent infections between viruses and parasites is a common feature in immunocompromised patients and especially during AIDS.(ABSTRACT TRUNCATED AT 400 WORDS)

[Isolation of Toxoplasma gondii by cellular culture in HIV infected patients]. / Isolement de Toxoplasma gondii par culture cellulaire chez les sujets infectés par le VIH.

Nine hundred and eight biological samples obtained from HIV-infected patients were examined for Toxoplasma gondii by a tissue culture method. Sixty-two isolations of Toxoplasma gondii were made in 43 patients. Fourteen isolations were made from tissue biopsies (brain 4, striated muscle 2, liver 1), bone marrow aspirates (4 cases), pericardial or pleural effusions (3 cases) and vitreous body (1). In 6 cases, a parasitic dissemination was evidenced by the demonstration of Toxoplasma in blood or other tissues or body fluids. Toxoplasma organisms were demonstrated in the bronchoalveolar lavage fluid of 19 patients; this was associated with a parasitaemia in 6 of the 10 patients whose blood sample was cultured at the same time. Among 273 blood samples cultured, 29 were positive for Toxoplasma (24 patients); all patients with parasitaemia had clinical symptoms, but only 8 of them had clinical or radiological symptoms suggestive of a cerebral localization; this suggests that the presence of a parasitaemia is fairly indicative of an extra cerebral localization of toxoplasmosis. In 11 patients, blood was examined after treatment was initiated; in 8 patients parasitaemia was undetectable at the first examination after treatment (1 to 15 days after initiation of therapy), whereas in 3 patients sampled 1, 4 or 6 days respectively after treatment a persistent parasitaemia could be demonstrated. These results clearly show that the tissue culture method is efficient and sensitive to provide an early evidence of infection (4 days), and may prove useful to evaluate therapeutic effectiveness.

Evaluation of a new 5'-nuclease real-time PCR assay targeting the Toxoplasma gondii AF146527 genomic repeat.

Toxoplasma gondii can be responsible for congenital toxoplasmosis leading to mild or severe sequelae, and for life-threatening infections in immunocompromised hosts. A new 5'-nuclease real-time PCR assay that targets the 300-fold repeated AF146527 DNA sequence (TaqMan-AF-PCR) has been developed and its performance for diagnosis of toxoplasmosis and treatment follow-up has been assessed. A retrospective analysis was first performed with 144 clinical specimens previously analysed for the presence of T. gondii DNA by a PCR-ELISA assay that targets the B1 gene of T. gondii (B1-PCR-ELISA). Fifteen samples, all from patients with clinically proven toxoplasmosis, were negative according to B1-PCR-ELISA and positive according to TaqMan-AF-PCR. A prospective analysis was then performed with 203 consecutive clinical specimens received at the laboratory of Parasitology of Saint-Louis Hospital during a 4-month period. The diagnosis of toxoplasmosis in two patients was made according to the TaqMan-AF-PCR whereas the B1-PCR-ELISA failed to make diagnosis. Additionally, iterative samples from a patient with cerebral and disseminated toxoplasmosis, already tested using a B1 real-time PCR assay, were tested using the TaqMan-AF-PCR and a Light Cycler real-time PCR assay targeting the same repetitive AF146527 sequence (LC-AF-PCR). Detection was achieved with the TaqMan-AF-PCR, with a mean gain of 7.1 and 3.3 amplification cycles when compared with the B1 real-time PCR and the LC-AF-PCR, respectively. This study demonstrates the higher sensitivity of the 5'-nuclease real-time PCR assay developed for the AF146527 DNA sequence and confirms the interest of using this highly repeated target to improve the diagnosis of toxoplasmosis.

[Severe disseminated toxoplasmosis with atypical chorioretinitis. A case of primary infection]. / Toxoplasmose disséminée sévère avec choriorétinite atypique : à propos d'un cas de primo-infection.

The clinical diagnosis of ocular toxoplasmosis is based on clinical features and biological tests: polymerase chain reaction (PCR) and the determination of intraocular specific antibody secretion (Goldmann-Witmer coefficient) on aqueous humor. Older patients may have a higher prevalence of ocular involvement and more severe ocular disease during the acute phase of recently acquired systemic infection because of altered cell-mediated immunity. Moreover, the genotype of the infecting parasite (particularly involving neotropical Type I Toxoplasma gondii strain), appears to be an important determinant of disease severity.

Toxoplasma gondii: blood and tissue kinetics during acute and chronic infections in mice.

Acute lethal infections were obtained in mice by intraperitoneal (IP) injection of 10(2) or 10(4) tachyzoites of the virulent RH and C56 strains. Chronic infections were obtained by IP injection or peroral (PO) gavage of 20 cysts of the avirulent C strain. Mice were sacrificed at varying intervals after infection and parasite burdens were quantitated in blood, brain, and lungs using a tissue culture method. Acutely infected mice died within 6 to 10 days postinfection as a function of the strain and inoculum size. With either strain, tachyzoites were first detected in lungs on either Days 2 or 4 postinfection, according to the inoculum size, then in brain and blood at Days 4 or 6; parasitic loads remained constantly at a higher level in lungs than in brain until the date of death. Bradyzoites could only be detected in lungs, from Days 4 or 6 until death. In chronic infections, similar results were obtained for IP and PO infected mice. Both tachyzoites and bradyzoites were first detected in lungs and brain from Day 7 after infection; tachyzoites remained at a higher level in lungs than in brain until Day 10, then subsequently decreased in lungs. At Day 50, tachyzoites were not detectable in lungs, whereas bradyzoites remained at a constant level; in brain, both parasitic stages were detectable at a similar level throughout the follow-up period. These results indicate that infection with a virulent Toxoplasma strain is characterized by an early involvement of lungs, with pneumonia as the principal cause of death.(ABSTRACT TRUNCATED AT 250 WORDS)

[Value and limitations of toxoplasmosis serology in HIV patients]. / Intérêt et limites de la sérologie de toxoplasmose chez les sujets VIH+.

Antibody titers to Toxoplasma gondii were studied in 62 AIDS patients with active toxoplasmosis (cerebral in 42, pulmonary in 10 and ocular in 10), confirmed by biopsy or by imaging techniques with a therapeutic test, and in 1,499 HIV-positive patients. The purpose of this study was to evaluate the value of antibody assays for the diagnosis of active infection and the prevalence of toxoplasmosis in HIV-positive individuals. IgG antibodies to Toxoplasma were found in 61 of the 62 AIDS patients, but not in one patient with pulmonary toxoplasmosis, with no significant differences in mean titers obtained by dye test, indirect immunofluorescence and sensitized agglutination. Twenty patients (31.7%) had dye test titers of 400 IU/ml or more; three patients had IgM antibodies. Thirteen (38%) of the 34 patients who had serial antibody assays exhibited a rise in IgG titers with no detectable production of IgM antibodies. Antibodies to Toxoplasma were found in 75% of the 1,499 HIV-positive subjects, a proportion which is not significantly different from that seen in HIV-negative controls; however, HIV-positive subjects were significantly more likely than controls to have high titers (greater than or equal to 500 IU in 18.7% of patients versus 9.2% of controls, p less than 0.001). A follow-up study in 177 HIV-positive patients with antibodies to Toxoplasma showed an annual reactivation rate of 12%; in five of 30 patients, the rise in antibody titers occurred concomitantly with or a few months before clinical toxoplasmosis; 25 patients remained asymptomatic.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of tissue culture and mouse inoculation methods for demonstration of Toxoplasma gondii.

Two methods for the isolation of Toxoplasma gondii were analyzed and compared. Bradyzoites or tachyzoites of three strains of T. gondii were injected into mice and introduced in parallel onto MRC5 fibroblasts cultured on cover slips. In the cultures, the parasites were more readily identified by an indirect immunofluorescence assay than by examination of unstained or Giemsa-stained cultures. With the RH strain, the tachyzoites replicated actively, and large foci of parasites were observed in 24 h. The bradyzoites or tachyzoites of the other strains could also be cultivated, but grew rather slowly; 2 days after inoculation, early stages of multiplication could be observed: from day +4, Toxoplasma clusters or foci were easily identified at a x100 magnification. The course of infection in mice was greatly dependent on the virulence of the strain and on the parasitic stage inoculated. In the chronically infected mice, evidence of Toxoplasma infection was only detected 45 days after inoculation through the demonstration of cysts in the brain or the presence of specific antibodies in the serum. The mean ratio of infected mice and positive cultures was compared in relation to the inoculum size. The tissue culture method was found to be at least as sensitive as mouse inoculation. Since Toxoplasma organisms may be isolated within a few days in tissue culture, it is proposed that this method should be used when early isolation of the parasite is crucial for the diagnosis of toxoplasmosis.