Odile Villard

Aspergillus galactomannan detection in the diagnosis of invasive aspergillosis in cancer patients.

PURPOSE To assess the Aspergillus galactomannan enzyme-linked immunosorbent assay (ELISA) in the diagnosis of invasive aspergillosis (IA) in adult and pediatric oncohematologic patients. PATIENTS AND METHODS The study was conducted in four patient groups: those with fever of unknown origin (FUO) during neutropenia, suspected pulmonary infection (PI), or nonpulmonary aspergillosis (NPA) and those undergoing surveillance (S) after hematopoietic stem-cell transplantation (HSCT). IA was classified as definite, probable, or possible, according to European Organization for Research and Treatment of Cancer/Mycosis Study Group definitions. RESULTS A total of 3,294 serum samples were collected during 797 episodes (FUO, 261; PI, 297; NPA, 28; and surveillance, 211), and 153 episodes of IA were diagnosed (31 definite, 67 probable, and 55 possible). Three episodes were first suspected from galactomannan ELISA; the remaining 150 cases were diagnosed from clinical or radiologic evidence. Sensitivity of the ELISA was 64.5%, 16.4%, and 25.5% in definite, probable, and possible episodes of IA, respectively, and was lower in patients positive for anti-Aspergillus antibodies than in antibody-negative patients. Most false-positive results occurred in children and in allogeneic HSCT (allo-HSCT) patients. Overall specificity of the ELISA was 94.8%. It was lower in children compared with adults (P <.0001) and in allo-HSCT patients compared with non-allo-HSCT adults (P =.0002). Lowering the ELISA cutoff value from 1.500 to 0.700 seemed more relevant for non-allo-HSCT adults (sensitivity, 73.1%, 44.3%, and 44.7% in definite, probable, and possible IA, respectively; specificity, 94%). CONCLUSION Galactomannan ELISA seems less sensitive than previously described, and sensitivity can be further reduced by the presence of anti-Aspergillus antibodies. A new cutoff value for the ELISA of 0.700 is proposed for non-allo-HSCT adults.

Comparison of Enzyme-Linked Immunosorbent Assay, Immunoblotting, and PCR for Diagnosis of Toxoplasmic Chorioretinitis

ABSTRACT Ocular toxoplasmosis is the major cause of posterior uvetis in European populations. The clinical diagnosis of toxoplasmic chorioretinitis is based upon ophthalmoscopic findings, which are often but not always typical. Laboratory testing is therefore important to confirm the etiology of the disease. In the present 2-year prospective study, the relative diagnostic sensitivities of the three analytical techniques (enzyme-linked immunosorbent assay [ELISA], immunoblotting, and PCR) were compared by using a group of patients (n = 19) with suspected ocular toxoplasmosis. The relative specificities of the three techniques were assessed by including two control groups of patients: one with nontoxoplasmic and noninflammatory ocular disease (n = 48) and the other with nontoxoplasmic and inflammatory ocular disease (n = 20). All 19 of the clinically suspect patients had serological evidence of exposure to Toxoplasma gondii: 17 had been previously infected, and 2 had current infection. The analysis of paired aqueous humor and serum samples by ELISA and immunoblotting revealed the local production of specific antibodies of the immunoglobulin G type in 63% (12 of 19) and 53% (10 of 19) of patients, respectively. PCR analysis of aqueous humor samples confirmed the presence of T. gondii DNA in 28% (5 of 18) of cases. When combined, ELISA, immunoblotting, and PCR findings confirmed the toxoplasmic origin of retinal lesions in 83% (15 of 18) of patients. The relative specificities of the three techniques were 89% for ELISA and immunoblotting and 100% for PCR.

Vaccination with Toxoplasma gondii SAG-1 Protein Is Protective against Congenital Toxoplasmosis in BALB/c Mice but Not in CBA/J Mice

ABSTRACT We evaluated the effect of vaccination with the SAG1 protein of Toxoplasma gondii against congenital toxoplasmosis in mice with different genetic backgrounds. In BALB/c mice (H-2d), vaccination reduced the number of infected fetuses by 50% and was associated with a mixed type 1 and type 2 immunity. In CBA/J mice (H-2k), vaccination increased the number of infected fetuses by 50% and was associated with a predominant type 2 response. Our results indicate that the effect of vaccination with SAG1 is controlled by the genetic background of the mouse.

Cytokine Profiles in Toxoplasmic and Viral Uveitis

BACKGROUND Uveitis is a major cause of visual impairment throughout the world. Analysis of cytokine profiles in aqueous humor specimens may provide insight into the physiopathological processes that underly retinal damage in this context. METHODS Using a multiplex assay, we determined the concentrations of 17 cytokines and chemokines in aqueous humor specimens obtained from patients with ocular toxoplasmosis or viral uveitis and compared these concentrations with those in specimens obtained from patients with noninfectious intermediate uveitis or cataract. RESULTS Five mediators (interleukin [IL]-8, monocyte chemoattractant protein-1, tumor necrosis factor-alpha, IL-4, and IL-10) were detected in >50% of patients in all groups. In contrast, IL-5 and IL-12 were specific for ocular toxoplasmosis, and granulocyte monocyte colony-stimulating factor and IL-1 were specific for viral uveitis; these mediators could present specific markers for diagnostic purposes. Interferon-gamma, IL-6, and macrophage inflammatory protein-1beta were common markers of ocular toxoplasmosis and viral uveitis. IL-17 was a common marker of ocular toxoplasmosis and intermediate uveitis. CONCLUSIONS We found specific cytokine profiles for each type of uveitis, with large interindividual variations and no etiological or clinical correlations. Ocular cytokine mapping contributes to a better understanding of the physiopathology of specific forms of uveitis and provides guidance for new targeted treatment.

Role of NK Cells and Gamma Interferon in Transplacental Passage of Toxoplasma gondii in a Mouse Model of Primary Infection

ABSTRACT Protective immunity in mice infected with Toxoplasma gondii is mainly mediated by NK cells, CD4 and CD8 T cells, and type 1 cytokines, such as gamma interferon (IFN-γ). To clarify the roles of NK cells and IFN-γ in protection against primary congenital toxoplasmosis, we used recombination activating gene 2 knockout (RAG-2−/−) mice, which lack T and B lymphocytes, in comparison with the wild-type BALB/c model. RAG-2−/− mice had a significantly lower risk of fetal toxoplasmosis than BALB/c mice (25 versus 63.9%; P = 0.003). This protection was associated with an increased number of maternal NK cells, IFN-γ secretion by spleen cells, and decreased parasitemia. In the RAG-2−/− mice, NK cell depletion increased both the rate of fetal infection, to 56.5% (P = 0.02), and the blood parasite burden. Conversely, in the BALB/c mice, this treatment did not modify maternofetal transmission or the blood parasite burden. Neutralization of IFN-γ in both infected RAG-2−/− and BALB/c mice decreased congenital Toxoplasma transmission, contrasting with an exacerbation of maternal infection. These data suggest that a partially protective immunity against congenital toxoplasmosis is achieved due to the increased number of NK cells in RAG-2−/− mice. However, it seems that IFN-γ enhances, directly or indirectly, the transplacental transmission.

Diagnosis of Congenital Toxoplasmosis: Comparison of Targets for Detection of Toxoplasma gondii by PCR

ABSTRACT Three PCR targets (18S ribosomal DNA, B1, and AF146527) and mouse inoculation were compared for 83 samples in the context of congenital toxoplasmosis. These four techniques are not statistically different in terms of sensitivity and specificity. However, further analysis highlighted problems sometimes encountered with PCR diagnosis of congenital toxoplasmosis.

Severe South American ocular toxoplasmosis is associated with decreased Ifn-γ/Il-17a and increased Il-6/Il-13 intraocular levels.

In a cross sectional study, 19 French and 23 Colombian cases of confirmed active ocular toxoplasmosis (OT) were evaluated. The objective was to compare clinical, parasitological and immunological responses and relate them to the infecting strains. A complete ocular examination was performed in each patient. The infecting strain was characterized by genotyping when intraocular Toxoplasma DNA was detectable, as well as by peptide-specific serotyping for each patient. To characterize the immune response, we assessed Toxoplasma protein recognition patterns by intraocular antibodies and the intraocular profile of cytokines, chemokines and growth factors. Significant differences were found for size of active lesions, unilateral macular involvement, unilateral visual impairment, vitreous inflammation, synechiae, and vasculitis, with higher values observed throughout for Colombian patients. Multilocus PCR-DNA sequence genotyping was only successful in three Colombian patients revealing one type I and two atypical strains. The Colombian OT patients possessed heterogeneous atypical serotypes whereas the French were uniformly reactive to type II strain peptides. The protein patterns recognized by intraocular antibodies and the cytokine patterns were strikingly different between the two populations. Intraocular IFN-γ and IL-17 expression was lower, while higher levels of IL-13 and IL-6 were detected in aqueous humor of Colombian patients. Our results are consistent with the hypothesis that South American strains may cause more severe OT due to an inhibition of the protective effect of IFN-γ.

Toxoplasma Seroconversion with Negative or Transient Immunoglobulin M in Pregnant Women: Myth or Reality? A French Multicenter Retrospective Study

ABSTRACT Classically, Toxoplasma infection is associated with high levels of specific IgM antibody and a rise in specific IgG levels 1 to 3 weeks later. Atypical IgG seroconversion, without IgM detection or with transient IgM levels, has been described during serologic follow-up of seronegative pregnant women and raises difficulties in interpreting the results. To evaluate the frequency and the characteristics of these atypical cases of seroconversion, an investigation was conducted within the French National Reference Center for Toxoplasmosis, from which 26 cases collected from 12 laboratories belonging to the network were identified. The aim of this work was to retrospectively analyze the results of serologic testing, the treatments administered, and the results of prenatal and postnatal follow-up for these women. In each case, IgG antibodies were detected using both screening and confirmatory tests. IgM antibodies were not detected in 15 cases, and the levels were equivocal or low-positive in 11 cases. The IgG avidity results were low in 16 cases and high in one case. Most of the pregnant women (22/26) were treated with spiramycin from the time that IgG antibodies appeared until delivery. Amniotic fluid was analyzed for Toxoplasma gondii DNA by PCR in 11/26 cases, and the results were negative in all cases. Congenital toxoplasmosis was ruled out in 12/26 newborns. There was no abnormality observed at birth for 10 newborns and no information available for 4 newborns. In conclusion, when the interpretation of serological results is so difficult, it seems cautious to initiate treatment by spiramycin and to follow the pregnant women and their newborns.

Comparison of Four Commercially Available Avidity Tests for Toxoplasma gondii-Specific IgG Antibodies

ABSTRACT Toxoplasma infection in pregnant women may cause congenital toxoplasmosis. Diagnosis of infection is based on serological tests aimed at detecting IgM and IgG antibodies against Toxoplasma gondii. However, IgM antibodies are not an accurate marker for discriminating between acute and latent infection. Detection of residual or persistent IgM may occur months or even years after primary infection, while the IgG avidity test is a rapid means of identifying latent infections in pregnant women who exhibit both IgG and IgM anti-Toxoplasma antibodies on initial testing during pregnancy. In this study, we assessed and compared the performances of four commercially available Toxoplasma IgG avidity tests in immunocompetent and immunocompromised patients with acute and latent toxoplasmosis. The positive predictive value of high avidity to confirm latent toxoplasmosis was 100% for all the assays, indicating that high avidity is a hallmark of latent infection. However, the negative predictive value of high avidity ranged from 99.2% (bioMérieux) to 95.3% (Abbott), indicating that acute toxoplasmosis could not be reliably diagnosed based on low IgG avidity alone. Thus, the avidity test provides a rapid means for identifying latent Toxoplasma infection in immunocompetent pregnant women presenting both IgG and IgM anti-Toxoplasma antibodies on initial testing. In terms of cost-effectiveness, avidity testing is a powerful tool that optimizes screening and follow-up of pregnant women while minimizing the costs of screening by avoiding subsequent costly maternal and fetal investigation and unnecessary treatment. The cheapest assay, Vidas Toxo IgG Avidity, also had the best performance for the diagnosis of latent toxoplasmosis.

Usefulness of PCR Analysis for Diagnosis of Alveolar Echinococcosis with Unusual Localizations: Two Case Studies

ABSTRACT The report presents two cases where diagnosis of alveolar echinococcosis was confirmed by Echinococcus multilocularis and Echinococcus granulosus PCR. The extrahepatic osseous involvement and the absence of initial hepatic involvement are unusual in both cases. Due to limitations of serological interpretation, PCR was useful to diagnose atypical echinococcosis.