Comparative performance of ISAGA IgM and ELISA assays for the diagnosis of maternal and congenital Toxoplasma infections: which technique could replace ISAGA IgM?

The ISAGA immunocapture test for the detection of anti-Toxoplasma immunoglobulin M is a manual technique known for its excellent sensitivity and specificity. The purpose of this retrospective, multicenter study was to compare the performances and agreement between ISAGA and other IgM detection techniques before cessation of ISAGA production. The analytic performance of the different tests was evaluated using 1,341 serum samples from adults with positive IgM and negative IgG to Toxoplasma gondii, and 1,206 sera from neonates born to mothers with seroconversion. The agreement between the tests was evaluated on 13,506 adult and 5,795 child serum samples. The sensitivity of Toxo-ISAGA IgM® (adults 98.7%, neonates 63.1%) was similar to that of Platelia Toxo IgM® (adults 94.4%, neonates 64.6%), and significantly higher than Liaison Toxo IgM® (adults 90.6%), Architect/Alinity Toxo IgM® (adults 95.7%, neonates 48.6%), and Vidas Toxo IgM® (adults 81.8%, neonates 17.5%). However, the specificities varied between 24.4% (Platelia Toxo IgM®) and 95.2% (Liaison Toxo IgM®) in adults and were >95% for all tests in neonates. An analysis of the kappa coefficients showed better agreement between ISAGA IgM® and the other tests in children (0.75-0.83%) than in adults (0.11-0.53%). We conclude that, in the absence of Toxo-ISAGA IgM®, the association of a very sensitive technique (Platelia Toxo IgM® or Architect/Alinity Toxo IgM®) and a very specific technique (Vidas Toxo IgM® or Liaison Toxo IgM®) is recommended for IgM detection in adult sera. For neonates, Platelia Toxo IgM® appeared to be the best alternative to replace Toxo-ISAGA IgM®.

Title: Performances comparatives des tests ISAGA IgM et ELISA pour le diagnostic des infections maternelles et congénitales à Toxoplasma : quelle technique pourrait remplacer ISAGA IgM ? Abstract: Le test d'immunocapture ISAGA pour la détection des immunoglobulines M anti-Toxoplasma est une technique manuelle connue pour son excellente sensibilité et spécificité. Le but de cette étude rétrospective et multicentrique était de comparer les performances et la concordance entre l'ISAGA et d'autres techniques de détection d'IgM avant l'arrêt de la commercialisation de l'ISAGA. Les performances analytiques des différents tests ont été évaluées à partir de 1 341 échantillons de sérum d'adultes présentant des IgM positives et des IgG négatives à Toxoplasma gondii, et de 1 206 sérums de nouveau-nés nés de mères présentant une séroconversion. La concordance entre les tests a été évaluée sur 13 506 échantillons de sérum d'adultes et 5 795 sérums d'enfants. La sensibilité de Toxo-ISAGA IgM® (adultes 98,7 %, nouveau-nés 63,1 %) était similaire à celle de Platelia Toxo IgM® (adultes 94,4 %, nouveau-nés 64,6 %) et significativement supérieure à celle de Liaison Toxo IgM® (adultes 90,6 %), Architect/Alinity Toxo IgM® (adultes 95,7 %, nouveau-nés 48,6 %) et Vidas Toxo IgM® (adultes 81,8 %, nouveau-nés 17,5 %). Cependant, les spécificités variaient entre 24,4 % (Platelia Toxo IgM®) et 95,2 % (Liaison Toxo IgM®) chez les adultes et étaient >95 % pour tous les tests chez les nouveau-nés. L'analyse des coefficients kappa a montré une meilleure concordance entre ISAGA IgM® et les autres tests chez les enfants (0,75­0,83%) que chez les adultes (0,11­0,53%). Nous concluons qu'en l'absence de Toxo-ISAGA IgM®, l'association d'une technique très sensible (Platelia Toxo IgM® ou Architect/Alinity Toxo IgM®) et d'une technique très spécifique (Vidas Toxo IgM® ou Liaison Toxo IgM®) est recommandée pour la détection des IgM dans les sérums adultes. Pour les nouveau-nés, Platelia Toxo IgM® apparaît comme la meilleure alternative en remplacement de Toxo-ISAGA IgM®.

Contribution of serology in congenital toxoplasmosis diagnosis: results from a 10-year French retrospective study.

This study aimed to evaluate different serological strategies for the postnatal diagnosis of congenital toxoplasmosis (CT) and establish a biological algorithm for CT diagnosis. The study analyzed serological data of immunoglobulins M, A, and G (IgM, IgA, IgG) performed by immunoenzymatic and compared immunological profile (CIP) assays in 668 newborns with CT diagnosis across four testing periods: P1 (D0- D10), P2 (D11-D35), P3 (D36-D45), and P4 (>D45). Forty-nine percent of the 668 CT cases were diagnosed during P1 and 34%, 4%, and 12% during P2, P3, and P4, respectively. CIP assays detected neosynthetized IgMs/IgGs in 98% of CT cases diagnosed during P1, while IgMs and IgAs were detected in 90% and 57% of CT cases diagnosed during P2 and in 88% and 67% of diagnoses made during P3, respectively. Detection of neosynthesized IgMs/IgGs, IgMs, and IgAs by immunoassay contributed to CT diagnosis in 81%, 77%, and 60% of cases, respectively. In total, 46% of serum samples were positive for all three parameters, 27% for two, and 27% for one of the three. The study recommends using the CIP assay as standard during P1 for CT diagnosis and IgM and IgA immunoassays after P1. A clinical and biological follow-up in a specialized center with a close collaboration between biologists and clinicians is highly recommended to increase the chances of early diagnosis. Overall, this study provides useful information for the development of a biological algorithm for CT diagnosis, which can aid in early detection and appropriate treatment of this disease.

IgM triplet in neonatal diagnosis by immunoblotting and its potential use as a diagnostic marker for congenital toxoplasmosis.

Primary infection during pregnancy by the protozoan Toxoplasma gondii can be worrisome because transmission to the fetus may lead to congenital toxoplasmosis (CT). Neonatal diagnosis is usually performed by serological profile comparison of the mother and newborn. As previously reported in 2012 by C. L'Ollivier et al., three IgM bands at 75, 90 and 100 kDa called the "IgM triplet" has caught our attention and seems to be pathognomonic of CT. This retrospective multicenter study involved nine reference laboratories included in the French National Reference Center for Toxoplasmosis network and concerned determining the specificity and sensitivity of this IgM triplet. On this basis, we were able to propose a new read of the comparison of IgG and IgM immunoblot profiles of mother and infant to increase the sensitivity of this diagnostic marker. The effect of the trimester of pregnancy at the time of infection, but also of maternal treatment with pyrimethamine/sulfadiazine/folinic acid on the presence of this IgM triplet in the infant, could be studied. The presence of the triplet appears pathognomonic for the diagnosis of CT, and it increased the sensitivity of the immunoblot assay from 55.04% to 72.48%. As a result, it would be wise to enhance conventional immunoblot reading by adding the presence of the three IgM bands in the infant pattern for neonatal diagnosis of CT.

Title: La triplette IgM dans le diagnostic néonatal par immunoblot et son utilisation potentielle comme marqueur diagnostique de la toxoplasmose congénitale. Abstract: La primo-infection pendant la grossesse par le protozoaire Toxoplasma gondii peut se révéler préoccupante car la transmission au fœtus peut conduire à une toxoplasmose congénitale (TC). Un diagnostic néonatal est généralement réalisé par comparaison des profils sérologiques de la mère et du nouveau-né. Comme précédemment rapporté en 2012 par C. L'Ollivier et al., l'association de trois bandes d'IgM à 75, 90, et 100 kDa appelée la « triplette IgM ¼ a retenu notre attention et semble être pathognomonique de la TC. Cette étude rétrospective multicentrique impliquant neuf laboratoires de référence inclus dans le réseau du Centre National de Référence pour la Toxoplasmose a permis de déterminer la spécificité et la sensibilité de cette triplette IgM. Ainsi, cela a permis de proposer une nouvelle lecture de la comparaison des profils d'immunoblot IgG et IgM de la mère et du nourrisson pour augmenter la sensibilité de ce marqueur diagnostique. L'effet du trimestre de la grossesse au moment de l'infection mais aussi du traitement maternel par pyriméthamine/sulfadiazine/acide folinique sur la présence de la triplette IgM chez l'enfant a pu être analysé. La présence de cette triplette semble pathognomonique pour le diagnostic de TC et elle permet d'augmenter la sensibilité du test immunoblot de 55,04 % à 72,48 %. Ainsi, il pourrait être judicieux d'améliorer la lecture conventionnelle de l'immunoblot en ajoutant la présence des trois bandes IgM dans le schéma du nourrisson pour le diagnostic néonatal de TC.

Advances in understanding and managing Scedosporium respiratory infections in patients with cystic fibrosis.

Introduction: Considered for a long time to be exclusively responsible for chronic localized infections, fungi of the genus Scedosporium have recently received a renewed interest because of their recognition as common colonizing agents of the respiratory tract of patients with cystic fibrosis, and of the description of severe disseminated infections in patients undergoing lung transplantation. Recently, several studies have been carried out on these opportunistic pathogens, which led to some advances in the understanding of their pathogenic mechanisms and in the biological diagnosis of the airway colonization/respiratory infections caused by these fungi.Areas covered: From a bibliographic search on the Pubmed database, we summarize the current knowledge about the taxonomy of Scedosporium species, the epidemiology of these fungi and their pathogenic mechanisms, and present the improvements in the detection of the airway colonization and diagnosis of Scedosporium respiratory infections, the difficulties in their therapeutic management, and the antifungal drugs in development.Expert opinion: As described in this review, many advances have been made regarding the taxonomy and ecology of Scedosporium species or the molecular determinants of their pathogenicity, but also in the management of Scedosporium infections, particularly by improving the biological diagnostic and publishing evidence for the efficacy of combined therapy.

Exophiala dermatitidis Revealing Cystic Fibrosis in Adult Patients with Chronic Pulmonary Disease.

Cystic fibrosis (CF) is a genetic inherited disease due to mutations in the gene cystic fibrosis transmembrane conductance regulator (CFTR). Because of the huge diversity of CFTR mutations, the CF phenotypes are highly heterogeneous, varying from typical to mild form of CF, also called atypical CF. These atypical features are more frequently diagnosed at adolescence or adulthood, and among clinical signs and symptoms leading to suspect a mild form of CF, colonization or infection of the respiratory tract due to well-known CF pathogens should be a warning signal. Exophiala dermatitidis is a melanized dimorphic fungus commonly detected in respiratory specimens from CF patients, but only very rarely from respiratory specimens from non-CF patients. We described here two cases of chronic colonization of the airways by E. dermatitidis, with recurrent pneumonia and hemoptysis in one patient, which led clinicians to diagnose mild forms of CF in these elderly patients who were 68- and 87-year-old. These cases of late CF diagnosis suggest that airway colonization or respiratory infections due to E. dermatitidis in patients with bronchiectasis should led to search for a mild form of CF, regardless of the age and associated symptoms. On a broader level, in patients with chronic respiratory disease and recurrent pulmonary infections, an allergic bronchopulmonary mycosis or an airway colonization by CF-related fungi like E. dermatitidis or some Aspergillus, Scedosporium or Rasamsonia species, should be considered as potential markers of atypical CF and should led clinicians to conduct investigations for CF diagnosis.

Purification and characterization of a mycelial catalase from Scedosporium boydii, a useful tool for specific antibody detection in patients with cystic fibrosis.

Scedosporium boydii is an opportunistic filamentous fungus which may be responsible for a wide variety of infections in immunocompetent and immunocompromised individuals. This fungus belongs to the Scedosporium apiospermum species complex, which usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF) and may lead to allergic bronchopulmonary mycoses, sensitization, or respiratory infections. Upon microbial infection, host phagocytic cells release reactive oxygen species (ROS), such as hydrogen peroxide, as part of the antimicrobial response. Catalases are known to protect pathogens against ROS by detoxification of the hydrogen peroxide. Here, we investigated the catalase equipment of Scedosporium boydii, one of the major pathogenic species in the S. apiospermum species complex. Three catalases were identified, and the mycelial catalase A1 was purified to homogeneity by a three-step chromatographic process. This enzyme is a monofunctional tetrameric protein of 460 kDa, consisting of four 82-kDa glycosylated subunits. The potential usefulness of this enzyme in serodiagnosis of S. apiospermum infections was then investigated by an enzyme-linked immunosorbent assay (ELISA), using 64 serum samples from CF patients. Whatever the species involved in the S. apiospermum complex, sera from infected patients were clearly differentiated from sera from patients with an Aspergillus fumigatus infection or those from CF patients without clinical and biological signs of a fungal infection and without any fungus recovered from sputum samples. These results suggest that catalase A1 is a good candidate for the development of an immunoassay for serodiagnosis of infections caused by the S. apiospermum complex in patients with CF.

Evaluation of the usefulness of six commercial agglutination assays for serologic diagnosis of toxoplasmosis.

Six agglutination tests for detecting Toxoplasma gondii-specific antibodies (immunoglobulin G or M) in serum were performed and compared. In total, 599 sera were examined using direct and indirect agglutination assays. Sensitivity varied from 93.7% to 100% and specificity from 97.1% to 99.2%. In a selected population with interfering diseases, the percentage of false positives ranged from 4.3% to 10.9%. Although an overall agreement of 100% was found for chronic toxoplasmosis, sensitivity for the detection of confirmed acute toxoplasmosis ranged from 86.4% to 97.3%. Regarding the large variability in terms of the performance of the 6 assays, tests based on the hemagglutination principle were found to be better than the other agglutination tests for all the panels evaluated, meaning that they could be used as qualitative or semiquantitative low-cost screening assays.

Occurrence and relevance of filamentous fungi in respiratory secretions of patients with cystic fibrosis--a review.

The colonization of airways by filamentous fungi and the development of respiratory infections require some predisposing factors as encountered in patients with cystic fibrosis (CF). Indeed, the defective mucociliary clearance which characterizes the disease is associated with local immunological disorders. In addition, the prolonged therapy with antibiotics and the use of corticosteroid treatments also facilitate fungal growth. An important fungal biota has been described in respiratory secretions of patients suffering from CF. Aspergillus fumigatus, Scedosporium apiospermum and Aspergillus terreus for filamentous fungi and Candida albicans for yeasts are the main fungal species associated with CF. Although less common, several fungal species including Aspergillus flavus and Aspergillus nidulans may be isolated transiently from CF respiratory secretions, while others such as Exophiala dermatitidis and Scedosporium prolificans may chronically colonize the airways. Moreover, some of them like Penicillium emersonii and Acrophialophora fusispora are encountered in humans almost exclusively in the context of CF. As fungal complications in CF patients are essentially caused by filamentous fungi the present review will not include works related to yeasts. In CF patients, fungi may sometimes be responsible for deterioration of lung function, as occurs in allergic broncho-pulmonary aspergillosis (ABPA) which is the most common fungal disease in this context. Additionally, although the clinical relevance of the fungal airway colonization is still a matter of debate, filamentous fungi may contribute to the local inflammatory response, and therefore to the progressive deterioration of the lung function.

Airway colonization by Acrophialophora fusispora in patients with cystic fibrosis.

Acrophialophora fusispora is a thermotolerant soil fungus which is very unusual in clinical samples. Here we report four cases of transient or chronic airway colonization by A. fusispora in patients with cystic fibrosis (CF). However, the prevalence of this fungus in CF patients may be underestimated due to the currently poor knowledge of this fungus in most of the medical mycology laboratories. In addition, its clinical importance regarding CF remains to be evaluated.

Genotyping study of Scedosporium apiospermum isolates from patients with cystic fibrosis.

Usually a saprophyte, Scedosporium apiospermum often colonizes the respiratory tracts of patients with cystic fibrosis (CF). In order to improve our understanding of the molecular epidemiology of the airway colonization, 129 sequential and multiple isolates collected from January 1998 to March 1999 from nine CF patients monitored in three hospitals in France were typed by random amplification of polymorphic DNA with primers GC70, UBC-701, and UBC-703. Among these primers, UBC-703 was the most discriminating, allowing the differentiation of 14 genotypes. Combining the results obtained with this three-primer set resulted in the differentiation of 16 genotypes. No common genotype was found among the different patients, and no clustering according to geographic origin of the isolates was seen. In addition, five of the patients were colonized by a single genotype. The others usually exhibited a predominant genotype accompanied by one or two others, which were found occasionally and were genetically close to the predominant genotype. Thus, our study demonstrates the persistence of the fungus despite antifungal treatments and therefore reinforces the need for the development of new antifungals that are more efficient against this species.

Typing of Scedosporium apiospermum by multilocus enzyme electrophoresis and random amplification of polymorphic DNA.

The genetic diversity among epidemiologically unrelated strains of the human pathogenic fungus Scedosporium apiospermum or its teleomorph, Pseudallescheria boydii, from different areas in Europe, was investigated by multilocus enzyme electrophoresis (MLEE) and random amplification of polymorphic DNA (RAPD). Fourteen enzyme activities were analysed by starch gel electrophoresis, corresponding to 27 polymorphic loci and 43 iso-enzymes. Among the enzymes studied, propionate esterase, carboxyl esterase, superoxide dismutase, carbonate dehydratase and malate dehydrogenase were the most polymorphic, allowing the classification of the strains into 6-11 groups each. Combination of the data obtained for the different enzyme activities studied allowed differentiation of the strains. Similarly, a high polymorphism was also revealed by each of the 20 RAPD primers tested, but no single primer was able to differentiate all the strains. The most efficient primers were GC70, UBC-701 and UBC-703, which revealed 17 distinct genotypes each, and combination of the results obtained with this three-primer set allowed complete discrimination of the strains. The dendrograms obtained from MLEE or RAPD by the unweighted pair-group method using arithmetic average cluster analysis did not reveal any clustering according to the geographic origin of the strains or their pathogenicity.

Molecular epidemiology of airway colonisation by Aspergillus fumigatus in cystic fibrosis patients.

A total of 109 sequential and multiple Aspergillus fumigatus isolates corresponding to 41 samples from seven cystic fibrosis (CF) patients was typed by random amplification of polymorphic DNA (RAPD) with the primer NS3 from the fungal ribosomal gene 18S subunit, and by sequence-specific DNA primer (SSDP) analysis. RAPD typing of the isolates revealed 10 different genotypes, whereas nine genotypes were identified by SSDP. Combination of the two typing methods permitted the differentiation of 25 overall genotypes. The colonisation typing patterns differed greatly between patients colonised for <1 year by A. fumigatus and long-term colonised patients. Two of three recently colonised patients presented a large number of types even in the same sample, unlike the chronically colonised patients, who harboured a limited number of genotypes. In the latter, the occurrence of a dominant genotype, usually the overall genotype 2, tended to reflect to the duration of colonisation. Moreover, anti-catalase antibodies to A. fumigatus appeared in most cases to be in response to genotype 2. These findings suggest that some strains of A. fumigatus may be selected during prolonged colonisation of the airways in CF patients.

Routine use of CHROMagar Candida medium for presumptive identification of Candida yeast species and detection of mixed fungal populations.

OBJECTIVE: To assess the value of the new differential culture medium CHROMagar Candida for routine investigation of clinical specimens. METHODS: During a whole year, 6150 clinical samples were plated on CHROMagar Candida medium. After incubation, the green colonies were considered to be Candida albicans. The colonies of other colors were identified using Bichrolatex-krusei, or by their assimilation pattern on ID 32C test strips and their morphology on rice cream-agar-Tween. RESULTS: Among the 6150 clinical samples, 1643 were positive for fungi. Aspergillus fumigatus and Geotrichum sp. were the predominant filamentous fungi isolated. Candida albicans was the most common species isolated (1274 of the positive samples; 77.5%), and Candida glabrata was the second most common yeast isolated (174 positive samples; 10.6%). Other yeast species were detected at lower frequencies, mainly Candida tropicalis (3.8%), Candida krusei (2.7%), Saccharomyces cerevisiae (2.7%) and Candida kefyr (2.3%), and 16 samples revealed a lipophilic species, Malassezia furfur. Mixed fungal populations accounted for 14.7% of the positive samples. Two or more yeast species were detected in 206 of the 242 specimens containing mixed fungal populations, and five yeast species were detected in one sample. Additionally, we did not observe significant differences in the isolation of yeasts or filamentous fungi from the 366 samples simultaneously plated on CHROMagar Candida and Sabouraud dextrose agar. Close agreement between the two culture media was observed for 89.9% of these samples. CONCLUSIONS: CHROMagar Candida medium was shown to be extremely helpful in a routine clinical mycology service, facilitating the detection of mixed cultures of yeasts and allowing direct identification of C. albicans, as well as rapid presumptive identification of the other yeasts: C. glabrata, C. tropicalis, C. krusei and S. cerevisiae. This chromogenic medium thus appears to be suitable as a primary culture medium, particularly for the mycologic surveillance of immunocompromised patients.