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.

[Prevalence of Candida parapsilosis, C. orthopsilosis and C. metapsilosis in candidemia over a 5-year period at Nantes hospital and in vitro susceptibility to three echinocandins by E-test®]. / Prévalence de Candida parapsilosis, C. orthopsilosis et de C. metapsilosis au sein des candidémies au CHU de Nantes et profil de sensibilité aux échinocandines par la méthode E-test®: étude rétrospective de cinq ans (2004-2009).

AIM OF THE STUDY: To determine the prevalence of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis among candidemia at Nantes University Hospital and to evaluate the in vitro susceptibility of the isolates against three echinocandin drugs (caspofungin, micafungin and anidulafungin). MATERIAL AND METHODS: Retrospective study (march 2004 to july 2009) of 178 cases of candidemia corresponding to 183 Candida spp. strains identified by means of routine phenotypical methods. Re-identification of C. parapsilosis sensu lato isolates was performed by ITS rDNA sequencing analysis. Minimal inhibitory concentrations (MIC) were determined by E-test(®). All echinocandin non-susceptible isolates (MIC>2 µg/mL) were analyzed for the presence/absence of FKS1 mutations associated with resistance. RESULTS: During this period, C. parapsilosis sensu lato was responsible for 27 candidemia, ranging at the second most common Candida species after C. albicans (n=99, 54.1%). Neither isolates belong to C. orthopsilosis nor C. metapsilosis. According to the literature, all the isolates displayed high MICs against the three echinocandin drugs. All the isolates displayed both susceptibility (MIC ≤ 2 µg/mL) and a good agreement between MICs read at 24h and 48 h for caspofungin and micafungin (MIC(50)=0.75 µg/mL, MIC(90)=1.5 µg/mL). Surprisingly, whereas most of the strains were susceptible to anidulafungin at 24h (MIC(50)=1 µg/mL, MIC(90)=1.5 µg/mL), 14 (52 %) displayed non-susceptibility, despite the lack of mutation associated with resistance on FKS1, when reading was performed at 48 h (MIC(50)=3 µg/mL, MIC(90)=12 µg/mL). CONCLUSION: Prevalence of C. orthopsilosis and C. metapsilosis in patients with candidemia is low at Nantes University Hospital. The difficulty encountered with MIC reading by E-test(®) are discussed.

[Field assessment of the new rapid diagnostic test Ebola eZYSCREEN®]. / Validation sur le terrain du nouveau test de diagnostic rapide Ebola eZYSCREEN®.

During the Ebola virus disease outbreak in West Africa in 2014, the World Health Organization has pointed out the need for rapid diagnostic tests (RDT) affordable, sensitive, specific, user-friendly, rapid, equipment-free, and deliverable. The rapid diagnostic test (Lateral Flow Assay) Ebola eZYSCREEN® was developed in this emergency frame using monoclonal antibodies against the envelope glycoprotein of the virus. Two distinct versions have been industrialized, one for whole-blood samples and the other for serum/plasma samples. Both versions have an analytical detection limit of 105 pfu/ml, the stability is at least 393 days at 30°C and 120 days at 45°C. The nonretrospective and independent validation study was carried out in the course of the outbreak in Conakry and at the Ebola Treatment Center of Coyah (Guinea) on 144 patients. In this study, the RDT showed a sensitivity of 65.3% and a specificity of 98.9% on whole blood, a sensitivity of 74.5% and a specificity of 100% on serum. Results from the whole-blood version must be analyzed with caution because of the delay between the blood collection and the completion of the tests, which was out of specification (3 days on average instead of 2 h). In contrast to laboratory tests, this easy to use field test does not require sophisticated instrumentation or even electricity and can contribute to the diagnostic chain of Ebola virus disease taking into account its benefits, high stability, and specificity but also its limit of sensitivity compared to laboratory techniques RT-qPCR (Real-Time reverse transcription Polymerase Chain Reaction), which remain the reference for the diagnosis of Ebola. The RDT Ebola eZYSCREEN® was granted EC IVD (IVD = In Vitro Diagnostic) marking.

Value of cerebrospinal fluid cytochemical examination for the diagnosis of congenital toxoplasmosis at birth in France.

BACKGROUND: Because of routine screening and treatment of pregnant women for Toxoplasma infection in France, most neonates born to mothers who seroconverted during pregnancy are either not infected or asymptomatic. Early diagnosis relies mainly on radiologic, ophthalmologic and biologic tests. Cerebrospinal fluid (CSF) cytochemical evaluation is one of several tests performed in parallel to increase the overall sensitivity of the diagnostic evaluation. Our goal was to assess the value of cytochemical examination and to confirm whether using a portion of available CSF for this analysis is legitimate. METHODS: The individual performance of each of the two cytochemical tests and their combined value when used in parallel were assessed. These findings were then compared with the anti-Toxoplasma IgM and IgA serum titers and the clinical, ophthalmologic and radiologic findings at birth. RESULTS: CSF cytochemical analysis was possible in only 52% of the 233 children in the study. Our results in 112 children indicated poor sensitivity estimates. There was no significant change in the posttest probability of infection compared with the pretest estimation of risk in cases of a negative finding. After a mean follow-up of 80 months there was no evidence that CSF cytochemistry helped predict the risk of sequelae. CONCLUSION: In our setting cytochemical examination did not significantly contribute to the diagnosis of congenital infection at birth. Because of the limited quantity of CSF available, we suggest the use of other methods with higher yield.

Ultrastructural aspects of Plasmodium falciparum-infected erythrocyte adherence to endothelial cells of Saimiri brain microvasculature.

We have recently shown that some squirrel monkeys (Saimiri sciureus) develop cerebral malaria when experimentally infected with asexual blood stage forms of different Plasmodium falciparum isolates. Since cerebral malaria is neither an inconsistent nor predictable event, several clones of endothelial cells isolated from the squirrel monkey brain microvasculature have been developed. Infected red blood cell (IRBC) adherence involved the knobs and direct membrane interactions through pseudopodes and microvilli on the Saimiri brain endothelial cell (SBEC) surface, similar to that observed with both brain microvascular endothelial cells from a patient who died of cerebral malaria and the rhesus monkey/P. coatneyi cerebral malaria model. The involvement of pseudopodes and microvilli increase the endothelial cell surface for the attachment of IRBCs; however, they are already present before the SBECs are exposed to IRBCs. With some SBEC phenotypes, embedding of IRBCs into the cytoplasma membrane of the endothelial cell was observed, resulting in an extremely close apposition of both SBEC and IRBC membranes during the adherence process. Once IRBCs are adherent, particularly for the embedding type, heterocellular communication-like structures between the cells become apparent. The upregulation of CD36 and intercellular adhesion molecule-1 by soluble recombinant (sr)-tumor necrosis factor-alpha or sr-interferon-gamma did not modify the IRBC interactions with SBECs at the ultrastructural level. The study shows further that the observed differences of IRBC adherence are due to unidentified phenotypic differences of SBECs rather than to a parasite isolate or particular endothelial cell receptor-associated phenomenon. Exploring P. falciparum IRBC cytoadherence in the squirrel monkey using a homologous physiologic target cell model in vitro should be useful for the evaluation of vaccine strategies and drugs to prevent human cerebral malaria.

Specificity of low anti-Toxoplasma IgG titers with IMx and AxSYM Toxo IgG assays.

Low antibody titers (3 to 6 IU/mL) detected by IMx and AxSYM Toxo IgG assays in serum samples from 264 pregnant women were confirmed by the dye test and a high-sensitivity agglutination test in, respectively, 98.5% and 95.6% of cases, attesting a premunition of these patients.

Flow cytometric quantification of Toxoplasma gondii cellular infection and replication.

The invasion and replication of Toxoplasma gondii are usually analyzed through either optical microscopy or incorporation of tritiated uracil. A new method has been developed using flow cytometric analysis to examine the entry and replication of T. gondii RH strain in Saimiri brain endothelial cells. After cell fixation and permeabilization using saponin, intracellular T. gondii were labeled with a monoclonal antibody against T. gondii SAG-1 (P30; the major cell-surface antigen) followed by fluorescein-conjugated rabbit anti-mouse IgG. The percentage of infected cells obtained using flow cytometry correlated directly with that obtained by UV light microscopy (r = 0.97). The mean fluorescence intensity of infected cells reflects intracellular P30 and assesses intracellular replication. The distribution of fluorescence per infected cell, considered with the percentage of infected cells, also allows a qualitative analysis of replication. Such a method is rapid, easy, and does not require specialized equipment for radioactive labeling.

[Contribution of molecular biology and Aspergillus galactomannan antigen assay for the diagnosis of histoplasmosis]. / Contribution de la biologie moléculaire et de l'antigénémie galactomannane aspergillaire au diagnostic de l'histoplasmose.

We report a case of a pulmonary histoplasmosis in an HIV-positive patient usually living in Cambodia, with a positive Aspergillus galactomannan antigenemia resulting from a cross-reaction, that decreased after antifungal therapy. We discuss the potential interest of the detection of fungal DNA by PCR and Aspergillus galactomannan antigenemia for the diagnosis of histoplasmosis, especially in countries where Histoplasma capsulatum antigen testing is not available.

[Epidemiology of candidemia: a one-year prospective observational study in the west of France]. / Epidémiologie des candidémies: étude observationnelle prospective d'un an dans l'Ouest de la France.

OBJECTIVE: A one-year prospective, observational study was conducted in the west of France, to evaluate the epidemiology of candidemia. METHOD: During the year 2004, each patient with at least one blood culture yielding Candida sp. was included. For each episode of candidemia, mycological, demographical, clinical, and therapeutic data, as well as outcome, were collected. RESULTS: One hundred and ninety-three strains of Candida sp. were isolated in 186 patients, Candida albicans accounting for 54.9%, Candida glabrata for 18.7%, Candida parapsilosis for 12.9%, Candida tropicalis for 4.7% and Candida krusei for 4.1% of these isolates. A percentage of 84% of the Candida isolates were fully susceptible to fluconazole in vitro. Dose-dependent susceptibility or resistance to fluconazole was detected in more than one third of the Candida glabrata strains, of which 36% were also resistant to voriconazole. Two-thirds of the patients were males, and the mean age was 61.5 years. A percentage of 37% of patients were hospitalized in intensive care units. The main predisposing factors for candidemia were broad-spectrum antibiotics (75.8%), central venous catheter (72.6%), cancer or hematologic malignancy (47.3%), recent surgery (42.5%), total parenteral nutrition (37.6%). One hundred and fifty-four patients were treated with antifungal therapy, two-thirds of whom received fluconazole as first-line agent. Mortality was 49% overall, and was significantly higher in case of septic shock, advanced age, and absence of catheter removal.

Diagnosis of congenital toxoplasmosis at birth: what is the value of testing for IgM and IgA?

UNLABELLED: Recommendations vary on the best combination of tests to use for the diagnosis of subclinical congenital toxoplasmosis at birth. The diagnostic accuracy of IgM and IgA tests was assessed in the context of routine clinical practice on 233 newborns with congenital toxoplasmosis and 661 healthy controls. IgM/IgA sensibility and specificity were compared in cord and postnatal samples. Both tests were considerably more specific in neonatal blood (IgM: 98%; IgA: 100%) than in cordblood (IgM: 85%; IgA: 88%). Sensitivity for IgM and IgA was not significantly different in neonatal blood (61% and 60%, respectively) and cord blood (67% and 54%, respectively). Combining IgM and IgA increased the overall sensitivity to 73% without any significant loss in specificity (98%). The influence of the date of maternal infection on the sensitivity and negative predictive value was also clearly demonstrated. CONCLUSION: Because of their relatively low cost compared to more sophisticated methods, IgM and IgA tests should remain the main method for the routine diagnosis of congenital toxoplasmosis although follow up is essential to identify the Ca. 25% of infected children who are missed at birth on the basis of these tests.

A rapid flow cytometric method to explore cellular immunity against Toxoplasma gondii in humans.

To assess cell-mediated immunity to Toxoplasma gondii, we evaluated the expression of the activation antigens CD69, CD71, and CD25 on T lymphocytes by flow cytometry after specific in vitro stimulation of whole blood from 127 T. gondii-positive and 63 T. gondii-negative patients. T lymphocytes from many seropositive individuals did not express CD69 at 24 h after T. gondii antigen stimulation, but CD71 and CD25 were easily detectable on T cells from seropositive individuals 7 days after specific activation. CD25 was mainly expressed by stimulated CD4(+) T cells, and its detection on total T cells was both a sensitive (98%) and a specific (97%) indicator of prior T. gondii infection. These results make flow cytometric detection of CD25 an excellent candidate for screening cell-mediated immunity to T. gondii in vitro and an interesting tool for the diagnosis of congenital infection.

Cellular immunity to Toxoplasma gondii in congenitally infected newborns and immunocompetent infected hosts.

The aim of this study was to determine the frequency of anergy to Toxoplasma gondii in congenitally infected newborns and immunocompetent infected individuals. Specific anergy to Toxoplasma has been reported previously, especially in cases of congenital toxoplasmosis. Whole blood from 592 immunocompetent patients with suspected toxoplasmosis was cultured in the presence of soluble Toxoplasma antigen for 7 days. Activated T lymphocytes were detected by flow cytometry. In patients over 1 year of age, the percentage of soluble Toxoplasma antigen-stimulated T cells expressing the interleukin-2 receptor CD25 was higher in infected patients than in uninfected subjects (40.0+/-18.3% vs. 1.8+/-2.0%, P<0.0001). No differences were detected between seroconverters, i.e. those with recent rises in IgM and IgG antibodies, and those with acquired or congenital toxoplasmosis. Similar results were observed when congenitally infected ( n=38) and uninfected ( n=89) children under 1 year of age were compared (17.6+/-9.2% vs. 3.0+/-4.9%, P<0.0001). The sensitivity and specificity values of CD25 detection for diagnosis of congenital toxoplasmosis in infants were 95% and 89%, respectively, at a threshold value of 7% above control culture. The results show that specific cellular immunity is detectable in virtually all Toxoplasma-infected patients, including newborns. Detection of CD25 constitutes a simple, sensitive and specific test for diagnosis of congenital toxoplasmosis.