Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

BACKGROUND: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. METHODS: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. RESULTS: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. CONCLUSION: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis

Bilan de l'utilisation en pratique pneumologique de la tomographie par émission de positons au 18F-fluoro-déoxyglucose (à propos de treize cas pris en charge au centre hospitalier de Thonon-les-Bains)

Étude de 13 patients pris en charge entre décembre 2000 et mars 2003 et ayant bénéficié d'une exploration par tomographie par émission de positons au 18F-fluoro-déoxyglucose (TEP18FDG) dans le cadre d'anomalies pulmonaires. La TEP18FDG a permis d'éviter l'utilisation de techniques diagnostiques invasives dans les deux cas de nodules pulmonaires solitaires. Elle a permis de rectifier le stade des cancers initialement établi sur les seules données morphologiques. Elle a apporté une aide décisionnelle quant aux différentes options thérapeutiques. Elle a permis dans les sept cas pour lesquels la chirurgie d'exérèse pouvait s'envisager, d'affirmer ou non son utilité en fonction des stades de la maladie. La TEP18FDG est une technique d'imagerie utile aux patients ainsi qu'aux praticiens en raison de ses capacités à modifier les stades des cancers pulmonaires comme cela apparaît clairement ici, ce qui modifie les conduites thérapeutiques. Sa grande spécificité dans le staging ganglionnaire a été bien retrouvée. Elle possède également une potentialité sous-estimée dans la recherche précoce des localisations métastatiques et des récidives.GRENOBLE1-BU Médecine pharm. (385162101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Microsporum praecox: molecular identification of a new case and review of the literature

International audienceWe report a rare case of dermatophytosis due to Microsporum praecox in a 28-year-old female horse rider. The skin lesion was located on the right external malleolus. Microscopic examination of skin scrapings revealed a dermatophyte which was also isolated in culture. The identification of M. praecox was confirmed by molecular biology (sequence analysis of PCR products amplified from internal transcribed spacer regions with universal primers). Combined antifungal therapy with oral terbinafine and topical cyclopiroxolamide resulted in complete remission of the fungal lesion within 1 month. Since 1944, only 29 cases of human M. praecox infection have been reported in the literature. The clinical features and treatment of these cases are reviewed. The prevalence of M. praecox infection is probably underestimated, and systematic molecular identification could improve our understanding of the epidemiology of this fungal dermatosis

Prevalence of antibodies to Neospora caninum in horses in France

International audienc

Prevention of severe Candida infections in nonneutropenic, high-risk, critically ill patients: a randomized, double-blind, placebo-controlled trial in patients treated by selective digestive decontamination

OBJECTIVE: Infections caused by Candida spp. are a major cause of morbidity and mortality in critically ill patients and usually develop from endogenous colonization. We assessed the effectiveness of adding fluconazole to a selective digestive decontamination regimen to prevent candidal infections. DESIGN AND SETTING: We performed a prospective, randomized, double-blind, placebo-controlled trial among medical and surgical intensive care unit patients at a large university hospital. PATIENTS: All adult patients mechanically ventilated for at least 48 h with an expectation to remain so for at least an additional 72 h, and receiving selective decontamination of the digestive tract. INTERVENTIONS: Patients were randomly assigned fluconazole 100 mg daily (n=103) or placebo (n=101). MEASUREMENTS AND RESULTS: Candida infections occurred less frequently in the fluconazole group (5.8%) than in the placebo group (16%; rate ratio 0.35; Cl(95) 0.11-0.94). Some 90% of candidemia episodes occurred in the placebo group (rate ratio for fluconazole use 0.10; Cl(95) 0.02-0.74). The rate of treatment failure, development of candidal infection, or increased colonization, was 32% in the fluconazole group and 67% in the placebo group (P<0.001). Crude in-hospital mortality was similar in the two groups (39% fluconazole vs. 41% placebo). CONCLUSIONS: Prophylactic use of fluconazole in a selected group of mechanically ventilated patients at high risk for infection reduces the incidence of Candida infections, in particular candidemia

New Vidas assay for Toxoplasma-specific IgG avidity: evaluation on 603 sera.

After the development of the new version of the test Vidas Toxo IgG with antigens obtained from tachyzoites cultured on cells, a Vidas avidity test has been recently developed. The aim of this study was to assess the value of the determination of avidity on the new Vidas test. This avidity test was performed on 553 sera obtained from pregnant women whose dates of infection had been determined and on 50 sera obtained from immunosuppressed patients. In the group of infection occurring less than 4 months before sampling, the avidity index was 0.3 for 44/46 sera of pregnant women and for 47/47 sera of immunosuppressed patients. Thus, the new version of avidity test was helpful primarily to rule out that an infection had occurred within the prior 4 months

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France

International audienceNeospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection

Comparison of PCR-Enzyme-Linked Immunosorbent Assay and Real-Time PCR Assay for Diagnosis of an Unusual Case of Cerebral Toxoplasmosis in a Stem Cell Transplant Recipient

A PCR-enzyme-linked immunosorbent assay and a real-time PCR assay were compared for diagnosis and follow-up of cerebral toxoplasmosis in a stem cell transplant recipient. The sensitivity of detection was similar for both assays but was higher when the assays were performed on buffy coat rather than on whole blood or serum