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).

[Management of ocular toxoplasmosis in France: Results of a modified Delphi study]. / Prise en charge de la toxoplasmose oculaire en France : résultats d'une étude Delphi modifiée.

OBJECTIVE: To evaluate diagnostic and therapeutic practices and then establish a consensus on the management of ocular toxoplasmosis in France through a Delphi study. MATERIALS AND METHODS: Twenty-three French experts in ocular toxoplasmosis were invited to respond to a modified Delphi study conducted online, in the form of two questionnaires, in an attempt to establish a consensus on the diagnosis and management of this pathology. The threshold for identical responses to reach consensus was set at 70 %. RESULTS: The responses of 19 experts out of the 23 selected were obtained on the first questionnaire and 16 experts on the second. The main elements agreed upon by the experts were to treat patients with a decrease in visual acuity or an infectious focus within the posterior pole, to treat peripheral lesions only in the presence of significant inflammation, the prescription of first-line treatment with pyrimethamine-azithromycin, the use of corticosteroid therapy after a period of 24 to 48hours, the prophylaxis of frequent recurrences (more than 2 episodes per year) with trimethoprim-sulfamethoxazole as well as the implementation of prophylactic treatment of recurrences in immunocompromised patients. On the other hand, no consensus emerged with regard to the examinations to be carried out for the etiological diagnosis (anterior chamber paracentesis, fluorescein angiography, serology, etc.), second-line treatment (in the case of failure of first-line treatment), or treatment of peripheral foci. CONCLUSION: This study lays the foundations for possible randomized scientific studies to be conducted to clarify the management of ocular toxoplasmosis, on the one hand to confirm consensual clinical practices and on the other hand to guide practices for which no formal consensus has been demonstrated.

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.

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.

Interpretation of the Elecsys Toxo IgG avidity results for very low and very high index: study on 741 sera with a determined date of toxoplasmosis.

Initial results with the Elecsys Toxo IgG Avidity assay showed some potential for interpretation of a very low or very high index result. We aimed to examine these new insights into interpretation using a large panel of serum samples and to define the optimal thresholds. A total of 741 patient serum samples with known date of infection (from a few weeks to more than 9 months after infection), were analysed with the Elecsys Toxo IgG Avidity assay. Values ≥80% (threshold defined by the manufacturer) were reported in 289 sera; 288 sera were sampled more than 4 months after infection. Thus, avidity values ≥80% excluded an infection less than 4 months. Avidity values ≥90% were reported in 112 sera sampled more than 9 months after infection. Thus, avidity values ≥90% excluded infection less than 9 months. Moreover avidity values ≤15% were reported in the 62 sera sampled less than 3 months after infection. Thus avidity values ≤15% excluded infection more than 3 months.

Help in the Choice of Automated or Semiautomated Immunoassays for Serological Diagnosis of Toxoplasmosis: Evaluation of Nine Immunoassays by the French National Reference Center for Toxoplasmosis.

Toxoplasmosis, a benign infection, is asymptomatic or paucisymptomatic in over 80% of cases, except in immunocompetent patients suffering from ocular toxoplasmosis or in immunocompromised patients with opportunistic or congenital toxoplasmosis. Diagnosis is based mainly on serology testing. Thus, we compared the performance of the nine most commonly used commercial automated or semiautomated immunoassays for IgG and IgM Toxoplasma gondii antibody detection, that is, the Advia Centaur, Architect, AxSYM, Elecsys, Enzygnost, Liaison, Platelia, VIDAS, and VIDIA assays. The assays were conducted on four panels of serum samples derived during routine testing from patients with an interfering disease and who exhibited a low IgG antibody level in one of two clinical settings, namely, acute or chronic toxoplasmosis. As a result, IgG sensitivities ranged from 97.1% to 100%, and IgG specificities ranged from 99.5% to 100%. For IgG quantification, strong differences in IgG titers (expressed in IU/ml) were noted depending on the assay used. IgM sensitivities ranged from 65% to 97.9%, and IgM specificities ranged from 92.6% to 100%. For defining the best serological strategies to be implemented, it appears crucial to compare the diagnostic performance of the different tests with respect to their specificity and sensitivity in detecting the presence of IgG and IgM antibodies.

Serological diagnosis of Toxoplasma gondii infection: Recommendations from the French National Reference Center for Toxoplasmosis.

Toxoplasmosis manifests no clinical signs in 80% of cases in immunocompetent patient, causing immunization characterized by the persistence of cysts, particularly in brain, muscles, and retina. Assessing the serological status, based on testing for serum toxoplasma IgG and IgM antibodies, is essential in cases that are increasingly at risk for the more severe disease forms, such as congenital or ocular toxoplasmosis. This disease also exposes immunosuppressed patients to reactivation, which can lead to more widespread forms and increased mortality. By interpreting the serological results, we can estimate the risk of contamination or reactivation and define appropriate prophylactic and preventive measures, such as hygienic and dietetic, therapeutic, biological, and clinical follow-up, according to the clinical context. We hereby propose practical approaches based on serological data, resulting from a consensus of a group of experts from the French National Reference Center Network for Toxoplasmosis, according to both routine and specific clinical situations.

Superficial Fungal Infections in a French Teaching Hospital in Grenoble Area: Retrospective Study on 5470 Samples from 2001 to 2011.

BACKGROUND: Superficial fungal infections are predominantly caused by dermatophytes, but the spectrum of species involved is depending on geographic areas and lifestyle. Only few studies have recently described the French epidemiology of these infections, especially dermatophytosis. OBJECTIVES: To determine the epidemiological situation of superficial fungal infections and the spectrum of dermatophytes in Grenoble area. PATIENTS/METHODS: A retrospective study of mycological laboratory records from January 2001 to December 2011 was carried out among patients with suspected fungal infections in the Grenoble University Hospital. Samples (skin scrapings, nail clippings and hair specimens) were collected, and mycological analyses were carried out by conventional methods. RESULTS: A total of 5470 samples collected from 3740 patients were analysed. Among the 1984 (36.3 %) positive cultures, dermatophytes were identified in 1348/1984 (67.9 %) samples, non-dermatophytes in 636/1984 (32.1 %) samples (yeasts 24.4 %, moulds 7.7 %). Toenails and feet were the most frequent localizations collected (2032 samples, 37.1 %, 1181 samples, 21.5 %). CONCLUSION: These data show the predominance (more than 92.6 %) of anthropophilic dermatophytes (Trichophyton rubrum, Trichophyton interdigitale and Trichophyton tonsurans). Trichophyton rubrum was the most commonly (78.6 %) isolated dermatophyte. Among zoophilic dermatophytes, Trichophyton verrucosum and Microsporum persicolor were regularly isolated.

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.

Prevalence of toxoplasmosis in France in 1998: is there a difference between men and women? At what age do children become infected?

BACKGROUND: The only national seroprevalence data currently available on toxoplasmosis in France are from the national perinatal surveys of pregnant women conducted in 1995 and 2003. These surveys are national, exhaustive and cross-sectional studies of all pregnant women who give birth in France during one specified week. These cross-sectional studies, conducted among women of childbearing age (defined as 18 to 45 years), showed a positive correlation between seroprevalence and age, with a significant regional disparity. This study was performed in order to compare the prevalence of toxoplasmosis antibodies in men and women in the 18-45 age group, to confirm regional variations and to estimate the seroprevalence of toxoplasmosis in France for different age groups, particularly among children and among adults aged over 45 years. METHODS: Serum samples from 2060 subjects were available from a national serum bank that was established in 1997 as part of a European study on vaccine preventable diseases. The sera were tested for IgG antibodies in 2008-2009, by ELISA test, at the laboratory of parasitology-mycology, CHU Grenoble. RESULTS: The seroprevalence for the population aged 1-64 years was 55.4%. Seroprevalence did not vary between the sexes, except among those aged over 45 years, where it was higher in men than in women. Toxoplasmosis seroprevalence varied significantly by regions for all ages. It increased with age and we noted a stronger increase in prevalence in adolescents (10-20 years) than in other age groups. CONCLUSION: This study showed that children have limited exposure to Toxoplasma gondii and that seroprevalence in men and women does not differ for the population aged 45 years and under. This study confirms the geographical disparity in prevalence in France that has been found in other studies in women of childbearing age. This disparity cannot be explained by different laboratory techniques, because sera were tested in a single laboratory. The study also raises the possibility of extrapolating seroprevalences from ENP to the general population and thus estimating the seroprevalence in the French population.

Toxoplasma seroconversion with negative or transient immunoglobulin M in pregnant women: myth or reality? A French multicenter retrospective study.

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.

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.

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.

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.

Performance of the Now Malaria rapid diagnostic test with returned travellers: a 2-year retrospective study in a French teaching hospital.

Malaria caused by Plasmodium falciparum remains the major life-threatening parasitic infection in the world. The number of cases in non-endemic countries continues to increase, and it is important that misdiagnosis of malaria should not occur, especially in non-immune travellers, because of the high risk of a fatal outcome. In a retrospective study of 399 sera, the Now Malaria rapid test was compared with the quantitative buffy coat (QBC) test and microbiological examination of thin blood films. Compared with the QBC test and thin blood films, the Now Malaria test had sensitivity and specificity values of 96.4% and 97%, respectively, for the detection of pure P. falciparum infection. A negative predictive value of 99.4% allows this test to be included in diagnostic strategies for patients presenting with clinical suspicion of malaria. Two false-negative results were associated with low levels of parasitaemia in the specimens. Thus, use of the Now Malaria test alone to detect P. falciparum infection in non-endemic countries could lead to misdiagnosis of malaria. This rapid diagnostic test should therefore be performed in association with another prompt traditional method such as examination of thin blood films.

Disseminated toxoplasmosis with pulmonary involvement after heart transplantation.

We report a case of pulmonary toxoplasmosis after heart transplant despite the prophylactic anti-toxoplasmic treatment that was given but was not sufficient to prevent toxoplasmosis. However, the patient survived thanks to early diagnosis confirmed by polymerase chain reaction on blood and by serological techniques, and early treatment.

Usefulness of Western blot in serological follow-up of newborns suspected of congenital toxoplasmosis.

The goal of the study reported here was to compare the results of Western blot with other serological methods for testing newborns suspected of having congenital toxoplasmosis. Western blot, enzyme-linked immunosorbent assay, immunoglobulin (Ig)M immunosorbent agglutination assay, and indirect immunofluorescence assay were performed on the sera of 126 neonates collected at birth and at 1 and 3 months of life. Western blot was more sensitive than IgM detection with the immunosorbent agglutination assay (82.6% vs. 69.6%), and the specificity of the two methods was 96.1% and 92.2%, respectively. Among the serological techniques tested, the combination of Western blot (IgG and IgM) with IgM immunosorbent agglutination assay achieved the greatest improvement in the sensitivity of early (postpartum) diagnosis of congenital toxoplasmosis.