We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.
bronchoalveolar lavage (BAL) specimens from 150 patients (19 HIV-infected and 131 non-HIV-infected patients) were evaluated for identification of Pneumocystis jiroveci (formerly known as Pneumocystis carinii f. sp. hominis) using staining techniques, conventional PCR (mtLSUrRNA gene) and real-time PCR (MSG gene). Test results were compared to Pneumocystis pneumonia (PCP) confirmed by typical clinical findings and response to treatment. Sensitivity and specificity of the techniques were 60 and 100 % for staining (where either one or both techniques were positive), 100 and 87 . 0 % for conventional PCR and 100 and 84 . 9 % for real-time PCR, respectively. The use of a concentration of 10 3 copies of DNA per capillary of BAL as a cut-off (determined by real-time PCR) increased specificity from 84 . 9 to 98 . 6 % without reducing the sensitivity of the technique. This technique is rapid (,3 h) and therefore of major interest in differentiating between asymptomatic carriage and PCP. A BAL specimen with ,10 3 copies per capillary of Pneumocystis-specific DNA is more likely to indicate a chronic carrier state, but in such cases follow-up is required to ensure that the patient is not in the early stage of an active PCP.
Natural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1β. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-β secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance.
This study demonstrates that DBSs collected under local conditions in resource-limited settings are suitable for the differed quantification of HIV-1 RNA.
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