Toxoplasma gondii, a plea for a thorough investigation of its oncogenic potential.

It is estimated that 30 % of the world's population harbours the parasite Toxoplasma gondii, particularly in the brain. Beyond its implication in potentially severe opportunistic or congenital infections, this persistence has long been considered as without consequence. However, certain data in animals and humans suggest that this carriage may be linked to various neuropsychiatric or neurodegenerative disorders. The hypothesis of a potential cerebral oncogenicity of the parasite is also emerging. In this personal view, we will present the epidemiological arguments in favour of an association between toxoplasmosis and cerebral malignancy, before considering the points that could underlie a potential causal link. More specifically, we will focus on the brain as the preferred location for T. gondii persistence and the propensity of this parasite to interfere with the apoptosis and cell cycle signalling pathways of their host cell.

Magnetically localized and wash-free fluorescence immunoassay (MLFIA): proof of concept and clinical applications.

Immunoassays are used for many applications in various markets, from clinical diagnostics to the food industry, generally relying on gold-standard ELISAs that are sensitive, robust, and cheap but also time-consuming and labour intensive. As an alternative, we propose here the magnetically localized and wash-free fluorescence immunoassay (MLFIA): a no-wash assay to directly measure a biomolecule concentration, without mixing nor washing steps. To do so, a fluorescence no-wash measurement is performed to generate a detectable signal. It consists of a differential measurement between the fluorescence of fluorophores bound to magnetic nanoparticles specifically captured by micro-magnets against the residual background fluorescence of unbound fluorophores. Targeted biomolecules (antibodies or antigens) are locally concentrated on micro-magnet lines, with the number of captured biomolecules quantitatively measured without any washing step. The performance of the MLFIA platform is assessed and its use is demonstrated with several biological models as well as clinical blood samples for HIV, HCV and HBV detection, with benchmarking to standard analyzers of healthcare laboratories. Thus, we demonstrated for the first time the versatility of the innovative MLFIA platform. We highlighted promising performances with the successful quantitative detection of various targets (antigens and antibodies), in different biological samples (serum and plasma), for different clinical tests (HCV, HBV, HIV).

Toxoplasma gondii-specific IgG avidity testing in pregnant women.

BACKGROUND: The parasite Toxoplasma gondii can cause congenital toxoplasmosis following primary infection in a pregnant woman. It is therefore important to distinguish between recent and past infection when both T. gondii-specific IgM and IgG are detected in a single serum in pregnant women. Toxoplasma gondii-specific IgG avidity testing is an essential tool to help to date the infection. However, interpretation of its results can be complex. OBJECTIVES: To review the benefits and limitations of T. gondii-specific avidity testing in pregnant women, to help practitioners to interpret the results and adapt the patient management. SOURCES: PubMed search with the keywords avidity, toxoplasmosis and Toxoplasma gondii for articles published from 1989 to 2019. CONTENT: Toxoplasma gondii-specific IgG avidity testing remains a key tool for dating a T. gondii infection in immunocompetent pregnant women. Several commercial assays are available and display comparable performances. A high avidity result obtained on a first-trimester serum sample is indicative of a past infection, which occurred before pregnancy. To date, a low avidity result must still be considered as non-informative to date the infection, although some authors suggest that very low avidity results are highly suggestive of recent infections depending on the assay. Interpretation of low or grey zone avidity results on a first-trimester serum sample, as well as any avidity result on a second-trimester or third-trimester serum sample, is more complex and requires recourse to expert toxoplasmosis laboratories. IMPLICATIONS: Although used for about 30 years, T. gondii-specific avidity testing has scarcely evolved. The same difficulties in interpretation have persisted over the years. Some authors have proposed additional thresholds to exclude an infection of <9 months, or in contrast to confirm a recent infection. Such thresholds would be of great interest to adapt management of pregnant women and avoid unnecessary treatment; however, they need confirmation and further studies.

Neonatal fever: A puzzling case.

Toxoplasmosis is a potentially serious fetal infection associated with maternal seroconversion of toxoplasmosis during pregnancy. Follow-up and treatment vary between different countries. We present a case of congenital toxoplasmosis with unusual physiopathology and symptomatology. The mother was immunized before the beginning of pregnancy but immunosuppressive treatments for Crohn disease maintained during the pregnancy could explain toxoplasmosis reactivation in the mother and congenital toxoplasmosis. The baby presented reversible B lymphopenia and hypogammaglobulinemia.

Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid.

OBJECTIVES: Molecular detection of Toxoplasma gondii plays a crucial role in the prenatal and neonatal diagnosis of congenital toxoplasmosis (CT). Sensitivity of this diagnosis is partly related to the efficiency of parasite DNA extraction and amplification. DNA extraction methods with automated platforms have been developed. Therefore, it is essential to evaluate them in combination with adequate PCR amplification assays. METHODS: In this multisite study, we investigated the suitability of two recent automated procedures for the isolation of Toxoplasma DNA from amniotic fluid (AF) (Magtration system 12GC, PSS and Freedom EVO VacS, Tecan), compared with three other automated procedures (MagNAPure Compact, Roche, BioRobot EZ1, Qiagen and modified NucliSens easyMAG, bioMérieux) and with the manual DNA extraction QIAamp DNA Mini kit (Qiagen). Two Toxoplasma PCR assays targeting the '529-bp' repeat DNA element were used, based upon dual hybridization (FRET) or hydrolysis (TaqMan) probes. A total of 1296 PCRs were performed including 972 Toxoplasma PCRs. RESULTS: We showed variable efficacy (4.2%-100% positive results) among the DNA extraction procedures in isolating up to five T. gondii cells/mL in AF samples. Moreover, for a given DNA extraction method, variable results were obtained among the two Toxoplasma PCR assays for detecting up to five T. gondii cells/mL: when using TaqMan PCR, all the automated systems yielded more than 60% positive results. Nevertheless, when testing the DNA extracts in triplicate, four out of six extraction methods allowed a satisfactory detection of low amounts of T. gondii DNA (≥33% of positive results) independently of the PCR assay used. CONCLUSIONS: Despite the influence of the subsequent PCR method used, this study should help microbiologists in the choice of DNA extraction methods for the detection of T. gondii in amniotic fluid. The extraction method should be checked as adequate for the PCR assay used.

[French recommendations on control measures to reduce the infectious risk in immunocompromised patients]. / Quelles mesures pour maîtriser le risque infectieux chez les patients immunodéprimés ? Recommandations formalisées d'experts.

The increase use of immunosuppressive treatments in patients with solid cancer and/or inflammatory diseases requires revisiting our practices for the prevention of infectious risk in the care setting. A review of the literature by a multidisciplinary working group at the beginning of 2014 wished to answer the following 4 questions to improve healthcare immunocompromised patients: (I) How can we define immunocompromised patients with high, intermediate and low infectious risk, (II) which air treatment should be recommended for this specific population? (III) What additional precautions should be recommended for immunocompromised patients at risk for infection? (IV) Which global environmental control should be recommended? Based on data from the literature and using the GRADE method, we propose 15 recommendations that could help to reduce the risk of infection in these exposed populations.

Evaluation of a new packaging process for non-autoclavable endoscopes: results for the first 100 microbiological samples.

BACKGROUND: A new process for packaging endoscopes (SureStore®, Medical Innovations Group) immediately after they exit from washing and disinfection in an automated endoscope reprocessor (AER) allows for endoscopes to be stored for up to 15 days. AIM: To describe the microbiological quality of samples from gastrointestinal endoscopes following this process. METHODS: Three-month prospective study using microbiological sampling from a stock of 38 gastrointestinal endoscopes carried out in a French University Hospital. The compliance rate (proportion of samples ≤25 cfu with no pathogenic micro-organisms) and the rate of sterile samples (proportion of germ-free samples) were calculated. We then used multivariate analysis to determine the factors associated with the maintenance of sterility. FINDINGS: One hundred samples were taken from stored endoscopes: 31 stored for ≤3 days, 34 stored between 3 and 7 days, and 35 after storage between 7 and 15 days. The compliance rate was 98% and the sterile sample rate was 60%. Only the time between leaving the AER and packaging was significantly associated with the sterility of samples (P = 0.02). The probability of having a sterile sample decreased 17-fold when the endoscope was packaged >2 h after leaving the AER (P = 0.04) compared to an endoscope packaged within 1 h after leaving the AER. CONCLUSION: The SureStore process seems capable of satisfactorily maintaining compliance (98%) of samples taken from endoscopes stored for up to 15 days. The delay in packaging should not exceed 1 h, as the rate of sterile samples decreases thereafter.

Long-term sera storage does not significantly modify the interpretation of toxoplasmosis serologies.

BACKGROUND: Serological investigation of Toxoplasma gondii can answer many questions about toxoplasmosis in human pathology. Along these lines, studies on serum storage in biobanks need to be performed especially in terms of determining the impact of storage on relevance of sera analysis after freezing. This study assessed the impact of long-term sera storage on the stability of anti-Toxoplasma immunoglobulins. MATERIAL AND METHODS: The stability of anti-Toxoplasma IgG and IgM was studied in 244 and 242 sera respectively, stored at -20°C from one month to ten years. ELISA-immunoassay (Vidas®, bioMérieux) was used for initial and post-storage analyses. Linear models for repeated measures and subgroup analyses were performed to assess the effect of storage duration and sample characteristics on immunoglobulins stability. RESULTS: Until ten years, the variability attributed to storage (maximum 8.07% for IgG, 13.17% for IgM) was below the variations inherent to the serological technique and allowed by quality assurance systems (15%). Subgroup analysis reported no variation attributed to sera storage. Serological interpretation was modified for 3 sera (1.2%) tested for IgM, all stored more than seven years. CONCLUSION: Anti-Toxoplasma immunoglobulins can reliably be measured for at least up to six years of storage with no modification of interpretation of toxoplasmosis serologies.

Prospective Evaluation of a New Aspergillus IgG Enzyme Immunoassay Kit for Diagnosis of Chronic and Allergic Pulmonary Aspergillosis.

Anti-Aspergillus IgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and Virion\Serion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens of Aspergillus fumigatus In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and Virion\Serion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P < 0.01), which was itself more sensitive than the Virion\Serion kit (P = 0.04). The Bordier and Bio-Rad kits had similar specificity (P = 0.8), both higher than that of the Virion\Serion kit (P = 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the Virion\Serion kits (0.977, 0.951, and 0.897, respectively; P < 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively; P = 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis.

En-suite bathrooms in protected haematology wards: a source of filamentous fungal contamination?

BACKGROUND: In spite of 25 recently built high-risk haematology rooms with a protected environment and fitted with en-suite bathrooms in our university hospital centre in 2008, sporadic cases of hospital-acquired invasive aspergillosis remained in these wards. AIM: This study aimed to identify unsuspected environmental sources of filamentous fungal contamination in these rooms. METHODS: Over two months, environmental fungal flora in the air (150 samples) as well as air particle counting and physical environmental parameters (airspeed, temperature, humidity, pressure) were prospectively monitored twice on the sampling day in all 25 protected rooms and en-suite bathrooms in use, and on bathroom surfaces (150 samples). FINDINGS: In rooms under laminar airflow, in the presence of patients during sampling sessions, fungi were isolated in two samples (4%, 2/50) with a maximum value of 2cfu/500L (none was Aspergillus sp.). However, 88% of the air samples (44/50) in the bathroom were contaminated with a median range and maximum value of 2 and 16cfu/500L. Aspergillus spp. were involved in 24% of contaminated samples (12/44) and A. fumigatus in 6% (3/44). Bathroom surfaces were contaminated by filamentous fungi in 5% of samples (8/150). CONCLUSION: This study highlighted that en-suite bathrooms in protected wards are likely to be a source of fungi. Before considering specific treatment of air in bathrooms, technicians have first corrected the identified deficiencies: replacement of high-efficiency particulate air filters, improvement of air control automation, and restoration of initial technical specifications. Assessment of measure effectiveness is planned.

Assessment of the IgA immunosorbent agglutination assay for the diagnosis of congenital toxoplasmosis on a series of 145 toxoplasmic seroconversions.

A retrospective analysis of 145 medical records from our teaching hospital laboratory showed an overall specificity of greater than 97% for the IgA immunosorbent agglutination assay (ISAGA A) performed on the sera of babies to diagnose congenital toxoplasmosis (CT). These actualized data emphasize the ability of this test to confirm a diagnosis of congenital toxoplasmosis.

Invasive aspergillosis: drug-dispensing systems as a source of filamentous fungal contamination in high-risk units?

High-risk units with air-control measures at Grenoble University Hospital are equipped with automated dispensing systems that are filled daily using drug trolleys routed from the pharmacy to the ward. The aim of this study was to evaluate the level of filamentous fungi (FF) contamination present in trolleys under usual conditions and after cleaning with Aniosurf(®) (fungicidal disinfectant). FF were detected in all samples, and 83.3% of samples were contaminated with Aspergillus fumigatus. Cleaning trolleys with Aniosurf(®) decreased the level of FF significantly, but contamination re-appeared within 24 h due to storage in a non-controlled environment.

Community-acquired invasive aspergillosis and outdoor filamentous fungal spore load: a relationship?

The daily number of outdoor spores was counted and the cases of community-acquired invasive aspergillosis (IA) were observed over a period of 31 months. The outdoor fungal load preceding IA occurrences was significantly higher than that measured during IA-free periods, underlining the importance of preventive measures to protect high-risk patients, even at home.

In vitro differential phenotypic characteristics among Type-II Toxoplasma gondii strains from congenital toxoplasmosis in humans.

In France, more than 80% of human congenital toxoplasmosis is attributable to Toxoplasma gondii strains belonging to the type-II lineage, whereas 3 main lineages have been described in Europe. Cell invasion, parasite replication, and cyst formation of type-II strains isolated from congenital infections were compared in a human cell model. The phenotype patterns of the 4 type-II strains studied differed from each other; the 2 strains responsible for asymptomatic infection formed significantly fewer cysts than the 2 strains isolated from symptomatic toxoplasmosis. The capacity to form cysts was different among the type-II strains studied and may be related to the clinical form.

[Results of 4 years of microbiological testing of bronchoscopes]. / Endoscopes bronchiques: bilan de quatre années de contrôles microbiologiques.

INTRODUCTION: In the context of reducing endoscopy-related infectious risk and new national guidelines on microbiological samples from bronchoscopy, the results of a surveillance program set up in a hospital center were analyzed. METHODS: Over 4 years, scheduled samples were taken from disinfected bronchoscopes. Bacteriology and mycology tests were used to search for microorganisms. The results were interpreted as falling within three levels: target, alert, and action. Factors that could explain the contamination were studied: age of the bronchoscope, number of uses per year, brand, and model. RESULTS: Out of 96 scheduled samples taken, the compliance rate for the period was 83% and increased (p=0.06) over the 4 years. We identified 15 Pseudomonas (six aeruginosa and nine other species), one Stenotrophomonas, one enterobacterium, and two filamentous fungi. None of the factors studied had a significant effect on sample contamination. CONCLUSION: The microbiological surveillance of bronchoscopes is an indispensable part of the quality assurance of bronchoscope maintenance. It can lead to maintenance of the bronchoscope when a noncompliant result is found.

Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control.

We have developed a quantitative PCR assay (LightCycler* using the pair of primers JW58 and JW59 for the detection of the 35-fold repeated B gene of oxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of . gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the same primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.106( )parasites in one ml of amniotic fluid (1 to 105( ) . gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.

[Real-time PCR in the diagnosis of toxoplasmosis: the way to standardisation?]. / Apport de la PCR quantitative dans le diagnostic de la toxoplasmose: la voie de la standardisation?

Severity of toxoplasmosis is highly correlated to the immune status of the infected individual. Foetus and immunocompromised patient are mostly at risk to develop life threatening forms. In this situation, serological diagnosis gives poor information. DNA detection using polymerase-chain-reaction technology (PCR) has significantly improved the management of this disease. Even so, the growing number of conventional PCR assays has finally led to variable performance results. Real-Time PCR (RT-PCR) in toxoplasmosis has been developed since 2000. This new technology can improve standardisation. Moreover, quantification of parasitic load in samples becomes possible. This review describes the main RT-PCR procedures actually under use and the studies comparing different target genes. The effective benefit of quantification is also discussed. Reducing number of procedures and more systematic external quality control should be considered, in order to improve reliability in PCR results, which has undoubtedly become a major tool in toxoplasmosis diagnosis.

Toxoplasma gondii in human astrocytes in vitro: interleukin (IL)-12 and IL-10 do not influence cystogenesis.

Interleukin (IL)-12, IL-10, and interferon (IFN)-gamma are major cytokines involved in the immune response against Toxoplasma gondii. Nevertheless, the role of IL-12 and IL-10 in the control of parasite replication and cytogenesis is not known yet, whereas the importance of IFN-gamma is documented. Furthermore, it is of paramount importance to study the interaction between T. gondii and cells from the central nervous system, e.g., astrocytes. In this study, we report that IL-12 and IL-10 have no effect on penetration, replication, or cystogenesis of the T. gondii Prugniaud strain in human astrocytes in vitro and do not antagonize the role of IFN-gamma on cystogenesis.

Ten-year surveillance of fungal contamination of food within a protected haematological unit.

Since 1992, we have established a protocol of food management (restrictive diet, food distribution protocol and fungal surveillance) for allogeneic stem-cell transplant (SCT) recipients hospitalised in protected ward. This study analyses the results of 10-year surveillance of fungal contamination of this diet. Among the 456 types of foods tested filamentous fungi were isolated in 37 of them (8.1%). Aspergillus fumigatus was isolated in one type of food only, while the majority of the food was contaminated to a lower extent.