Search

CRISPR-Cas is an adaptive immune system that prevents bacteria and archea from nucleic acids invasion such as viral genomes. The ability of the CRISPR-Cas technology to effectively and precisely cut a targeted genomic DNA region was exploited to develop powerful genome editing tools that were adapted for a wide range of applications, revolutionizing biological sciences. The CRISPR-Cas system consists of a Cas endonuclease triggered by a RNA guide for highly specific cleavage of targeted DNA or RNA sequences. In addition to the target specific cleavage, some Cas enzymes, including Cas12a and Cas13a, display a collateral trans-cleavage activity that allows the cleavage of all surrounding single-stranded nucleic acids. These biosensing activities of CRISPR-Cas systems, based on target specific binding and cleavage, are promising tools to develop accurate diagnostic methods to detect specific nucleic acids. CRISPRCas could therefore be used to diagnose a wide variety of diseases. In the current review we propose to describe the more significant advances for virus detection based on CRISPR-Cas systems.CRISPR-Cas est décrit comme un système immunitaire adaptatif qui permet aux bactéries et aux archées de se défendre contre les agressions virales. La technologie dérivée de ces systèmes CRISPR-Cas, qui permet de cliver précisément une séquence génomique, est désormais la base de puissants outils de biologie moléculaire et d’édition des génomes. Les « ciseaux moléculaires » CRISPR-Cas utilisent des endonucléases Cas, programmées et activées avec un ARN guide, pour couper spécifiquement une séquence cible ARN ou ADN. Certaines de ces enzymes Cas, notamment Cas12a et Cas13a, présentent au-delà de cette activité de coupure dirigée par un guide ARN, une activité générique de clivage collatéral en trans de toutes séquences nucléiques rencontrées. Ces différentes activités des systèmes CRISPR-Cas ont pu être exploitées pour développer des outils prometteurs de diagnostic moléculaire. Si les applications sont très nombreuses dans différents domaines, nous proposons ici d’illustrer le potentiel de ces approches basées sur CRISPR-Cas dans le cadre du diagnostic en virologie.

Human immunodeficiency virus (HIV) infection is a chronic disease that can be treated with antiretroviral (ARV) therapy. However, the success of this treatment has been jeopardized by the emergence of HIV infections resistant to ARV drugs. In low-to middle-income countries (LMICs), where transmission of resistant viruses has increased over the past decade, there is an urgent need to improve access to HIV drug resistance testing. Here, we present a proof-of-concept study of a rapid and simple molecular method to detect two major mutations (K103 N, Y181C) conferring resistance to first-line nonnucleoside reverse transcriptase inhibitor regimens. Our near-point-of-care (near-POC) diagnostic test, combining a sequence-specific primer extension and a lateral flow DNA microarray strip, allows visual detection of HIV drug resistance mutations (DRM) in a short turnaround time (4 h 30). The assay has a limit of detection of 100 copies of plasmid DNA and has a higher sensitivity than standard Sanger sequencing. The analytical performance was assessed by use of 16 plasma samples from individuals living with HIV-1 and results demonstrated the specificity and the sensitivity of this approach for multiplex detection of the two DRMs in a single test. Furthermore, this near-POC assay could be easily taylored to detect either new DRMs or DRM of from various HIV clades and might be useful for pre-therapy screening in LMICs with high levels of transmitted drug resistance.

International audience; Arbovirus diagnostics on blood from donors and travelers returning from endemic areas is increasingly important for better patient management and epidemiological surveillance. We developed a flexible approach based on a magnetic field-enhanced agglutination (MFEA) readout to detect either genomes or host-derived antibodies. Dengue viruses (DENVs) were selected as models. For genome detection, a pan-flavivirus amplification was performed before capture of biotinylated amplicons between magnetic nanoparticles (MNPs) grafted with DENV probes and anti-biotin antibodies. Magnetization cycles accelerated this chaining process to within 5 min while simple turbidimetry measured the signal. This molecular MFEA readout was evaluated on 43 DENV RNA(+) and 32 DENV RNA(−) samples previously screened by real-time RT-PCR. The sensitivity and the specificity were 88.37% (95% CI, 78.76%–97.95%) and 96.87% (95% CI, 90.84%–100%), respectively. For anti-DENV antibody detection, 103 plasma samples from donors were first screened using ELISA assays. An immunological MFEA readout was then performed by adding MNPs grafted with viral antigens to the samples. Anti-DENV antibodies were detected with a sensitivity and specificity of 90.62% (95% CI, 83.50%–97.76%) and 97.44% (95% CI, 92.48%–100%), respectively. This adaptable approach offers flexibility to platforms dedicated to the screening of emerging infections

Recent virus outbreaks have revealed a critical need for large scale serological assays. However, many available tests either require a cumbersome, costly apparatus or lack the availability of full automation. In order to address these limitations, we describe a homogeneous assay for antibody detection via measurement of superparamagnetic particles agglutination. Application of a magnetic field permits to overcome the limitations governed by Brownian translational diffusion in conventional assays and results in an important acceleration of the aggregation process as well as an improvement of the limit of detection. Furthermore, the use of protein-concentrated fluid such as 5 times-diluted human plasma does not impair the performances of the method. Screening of human plasma samples shows a strict discrimination between seropositive and seronegative samples in an assay duration as short as 14 s. The sensitivity of this method, combined with its quickness and simplicity, makes it a promising diagnostic tool.

International audience; The detection of DNA molecules by agglutination assays has suffered from a lack of specificity. The specificity can be improved by introducing a hybridization step with a specific probe. We developed a setting that captured biotinylated DNA targets between magnetic nanoparticles (MNPs) grafted with tetrathiolated probes and anti-biotin antibodies. The agglutination assay was enhanced using a series of magnetization cycles. This setting allowed to successfully detect a synthetic single stranded DNA with a sensitivity as low as 9 pM. We next adapted this setting to the detection of PCR products. We first developed an asymmetric pan-flavivirus amplification. Then, we demonstrated its ability to detect dengue virus with a limit of detection of 100 TCID 50 /mL. This magnetic field-enhanced agglutination assay is an endpoint readout, which benefits from the advantages of using nanoparticles that result in particular from a very reduced duration of the test; in our case it lasts less than 5 min. This approach provides a solution to develop new generation platforms for molecular diagnostics.

Creutzfeldt-Jakob diseases are neurodegenerative disorders for which transmission linked to medical procedures have been reported in hundreds of patients. As prion diseases, they are characterized by an unusual resistance to conventional decontamination processes. Moreover, their large tissue distribution and the ability of prions to attach to many surfaces raised the risk of transmission in health care facilities. It is therefore of major importance that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated for prion inactivation. We previously described an in vitro assay, which allowed us to classify accurately prion decontamination treatments according to their efficacy on variant Creutzfeldt-Jakob disease. The significance of this study is in demonstrating the concordance between previous in vitro results and infectivity studies in transgenic mice. Furthermore, commercial reagents currently used in hospitals were tested by both protocols, and we observed that most of them were ineffective on human prions., To date, approximately 500 iatrogenic Creutzfeldt-Jakob disease cases have been reported worldwide, most of them resulting from cadaveric dura mater graft and from the administration of prion-contaminated human growth hormone. The unusual resistance of prions to decontamination processes, their large tissue distribution, and the uncertainty about the prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the general population lead to specific recommendations regarding identification of tissue at risk and reprocessing of reusable medical devices, including the use of dedicated treatment for prion inactivation. We previously described an in vitro assay, called Surf-PMCA, which allowed us to classify prion decontamination treatments according to their efficacy on vCJD prions by monitoring residual seeding activity (RSA). Here, we used a transgenic mouse line permissive to vCJD prions to study the correlation between the RSA measured in vitro and the in vivo infectivity. Implantation in mouse brains of prion-contaminated steel wires subjected to different decontamination procedures allows us to demonstrate a good concordance between RSA measured by Surf-PMCA (in vitro) and residual infectivity (in vivo). These experiments emphasize the strength of the Surf-PMCA method as a rapid and sensitive assay for the evaluation of prion decontamination procedures and also confirm the lack of efficacy of several marketed reagents on vCJD prion decontamination. IMPORTANCE Creutzfeldt-Jakob diseases are neurodegenerative disorders for which transmission linked to medical procedures have been reported in hundreds of patients. As prion diseases, they are characterized by an unusual resistance to conventional decontamination processes. Moreover, their large tissue distribution and the ability of prions to attach to many surfaces raised the risk of transmission in health care facilities. It is therefore of major importance that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated for prion inactivation. We previously described an in vitro assay, which allowed us to classify accurately prion decontamination treatments according to their efficacy on variant Creutzfeldt-Jakob disease. The significance of this study is in demonstrating the concordance between previous in vitro results and infectivity studies in transgenic mice. Furthermore, commercial reagents currently used in hospitals were tested by both protocols, and we observed that most of them were ineffective on human prions.

L’emergence des arboviroses constitue une menace globale de sante publique compte tenu de la diffusion sur tous les continents des vecteurs et des virus associes a ces infections. La dengue, selectionnee comme modele, affecte annuellement plus de 390 millions de personnes dans le monde. Le manque de tests moleculaires rapides et facilement utilisables sur le terrain limite le diagnostic de l’infection et la prise en charge optimale des patients. Dans ce contexte, nous avons developpe une nouvelle approche analytique basee sur l’amplification isotherme des genomes viraux couplee a l’agglutination de nanoparticules magnetiques (NPs). Les genomes des virus Dengue (DENV1/2/3/4) sont amplifies et biotinyles par RT-RPA (Reverse Transcription Recombinase Polymerase Amplification) puis captures entre des NPs greffees par des sondes nucleiques et des NPs fonctionnalisees par des anticorps anti-biotine. L’application de cycles d’aimantation/relaxation permet d’accelerer cette capture et les agregats formes sont detectes en 5 min par une simple mesure d’absorbance evitant le recours a la fluorescence et a des instruments sophistiques. Une sensibilite analytique de 10 TCID50/mL a ete obtenue sur une gamme de reference (CNR Arboviroses). Sur 31 plasmas humains DENV depistes prealablement positifs en RT-qPCR (8 Fig. 1 ).

La forme variante de la maladie de Creutzfeldt-Jakob (vMCJ) est une maladie a prion, fatale, entrainant la destruction progressive des neurones. Elle resulte generalement de l’ingestion de viande bovine contaminee mais serait egalement transmissible par le sang. Malgre le declin de l’epidemie dans le monde, des incertitudes demeurent quant a la prevalence dans la population generale de porteurs asymptomatiques potentiellement capables de transmettre la maladie par transfusion sanguine ou par d’autres procedures medicales. Objectifs Evaluer l’existence de porteurs silencieux de l’infection vMCJ et la transmission transfusionnelle dans une population de patients polytransfuses. Methodes Nous avons publie le premier test permettant de detecter l’agent vMCJ dans des echantillons sanguins y compris au stade pre-symptomatique (Bougard et al., Sci Transl Med 2016). Ce test combine une capture des proteines prions sur des nanobilles et une etape d’amplification specifique du vMCJ afin de detecter des quantites infimes de prion dans le plasma. Nous avons analyse un panel de 100 echantillons issus de patients atteints d’anemie constitutionnelle, et multi-transfuses (en moy. 119 CGR recus) inclus dans la bio collection BOTIA (Blood and Organ Transmissible Infectious Agents) constituee de couples d’echantillons de donneurs/receveurs apparies avec un suivi longitudinal. Resultats Aucun des 100 echantillons de plasma analyses par notre test ultrasensible n’a ete detecte positif pour l’agent responsable de la vMCJ. Conclusions Ces resultats sont en faveur de la securite de la chaine transfusionnelle vis-a-vis du risque prion et seront a confirmer par des etudes a plus grande echelle.

The authors would like to express their sincere gratitude to Gaelle-Anne Cremer for furnishing the streptavidin-antibody conjugates, Patrice Sarfati and Pierre Sonigo for their expert supervision of the work, François Rieunier and Stephane Rihet for their pertinent advices on the work.; International audience; A nanoparticle-based electrochemical sandwich immunoassay was developed for bacteria detection in platelet concentrates. For the assay, magnetic beads were functionalized with antibodies to allow the specific capture of bacteria from the complex matrix, and innovative methylene blue-DNA/nanoparticle assemblies provided the electrochemical response for amplified detection. This nanoparticular system was designed as a temperature-sensitive nano-tool for electrochemical detection. First, oligonucleotide-functionalized nanoparticles were obtained by direct synthesis of the DNA strands on the nanoparticle surface using an automated oligonucleotide synthesizer. Densely packed DNA coverage was thus obtained. Then, DNA duplexes were constructed on the NP surface with a complementary strand bearing a 3 methylene blue tag. This strategy ultimately produced highly functionalized nanoparticles with electrochemical markers. These assemblies enabled amplification of the electrochemical signal, resulting in a very good sensitivity. A proof-of-concept was carried out for E. coli detection in human platelet concentrates. Bacterial contamination of this complex biological matrix is the highest residual infectious risk in blood transfusion. The development of a rapid assay that could reach 10–102 CFU mL−1 sensitivity is a great challenge. The nanoparticle-based electrochemical sandwich immunoassay carried out on a boron doped diamond electrode proved to be sensitive for E. coli detection in human platelets. Two antibody pairs were used to develop either a generic assay against certain Gram negative strains or a specific assay for E. coli. The methylene blue-DNA/nanoparticles amplify sensitivity ×1000 compared with the assay run without NPs for electrochemical detection. A limit of detection of 10 CFU mL−1 in a biological matrix was achieved for E. coli using the highly specific antibody pair.

There is a strong need of sensitive and accurate diagnostic tests of infectious prion proteins (PrPSc) in pre-symptomatic individuals and animals. This work associates, for the first time, the magnetic microparticle multimer detection system (MDS) with a recyclable boron doped diamond (BDD) electrode for the electrochemical detection of PrPSc in plasma samples from scrapie-infected sheep with clinical signs. The electrochemical results are in agreement with the chemiluminescence results. The main advantages of the electrochemical fluidic system are its recyclability and the use of low quantity of functional magnetic beads.

BACKGROUND Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. STUDY DESIGN AND METHODS A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. RESULTS Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. CONCLUSION In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

International audience; Nucleic acid testing during the preseroconversion viremic phase is required to differentially diagnose arboviral infections. The continuing emergence of arboviruses, such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV), necessitates the development of a flexible diagnostic approach. Similar clinical signs and the priority to protect pregnant women from ZIKV infection indicate that the differential diagnosis of arboviruses is essential for effective patient management, clinical care, and epidemiologic surveillance. We describe an innovative diagnostic approach that combines generic RT-PCR amplification and identification by hybridization to specific probes. Original tetrathiolated probes were designed for the robust, sensitive, and specific detection of amplified arboviral genomes. The limit of detection using cultured and quantified stocks of whole viruses was 1 TCID50/mL for DENV-1, DENV-3, and CHIKV and 10 TCID50/mL for DENV-2, DENV-4, and ZIKV. The assay had 100% specificity with no false-positive results. The approach was evaluated using 179 human samples that previously tested as positive for the presence of ZIKV, DENV, or CHIKV genomes. Accordingly, the diagnostic sensitivity for ZIKV, DENV, and CHIKV was 87.88% (n = 58/66), 96.67% (n = 58/60), and 94.34% (n = 50/53), respectively. This method could be easily adapted to include additional molecular targets. Moreover, this approach may also be adapted to develop highly specific, sensitive, and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses.

International audience; Conventional blood group phenotyping by hemagglutination assays, carried out pretransfusion, is unsuitable in certain clinical situations. Molecular typing offers an alternative method, allowing the deduction of blood group phenotype from genotype. However, current methods require a long turnaround time and are not performed on-site, limiting their application in emergency situations. Here, we report the development of a novel, rapid multiplex molecular method to identify seven alleles in three clinically relevant blood group systems (Kidd, Duffy and MNS). Our test, using a dry-reagent allele-specific lateral flow biosensor, does not require DNA extraction and allows easy visual determination of blood group genotype. Multiplex linear-after-the-exponential (LATE)-PCR and lateral flow parameters were optimized with a total processing time of one hour from receiving the blood sample. Our assay had a 100 % concordance rate between the deduced and the standard serological phenotype in a sample from 108 blood donors, showing the accuracy of the test. Owing to its simple handling, the assay can be operated by non-skilled health-care professionals. The proposed assay offers the potential for the development of other relevant single nucleotide polymorphism (SNP) panels for immunohematology and new applications, such as for infectious diseases, in the near future.

Le variant de la maladie de Creutzfeldt-Jakob (vMCJ) est une maladie a prion rare d’incubation longue et dont l’issue est toujours fatale. Malgre le declin de l’epidemie dans le monde, des incertitudes demeurent quant a la prevalence dans la population generale de porteurs asymptomatiques potentiellement capables de transmettre la maladie par transfusion sanguine ou par d’autres procedures medicales. Nous avons developpe un test de depistage suffisamment sensible pour identifier les patients vMCJ plus de 2 ans avant l’apparition des symptomes. Objectif Automatiser le test de detection du vMCJ dans le plasma. Methodes Le test comprend 3 etapes : – capture du prion dans le plasma a l’aide de nanobilles magnetiques recouverte de plasminogene ; – amplification in vitro du prion vMCJ par PMCA (Protein Misfolding Cyclic Amplification) ; – detection par Western-Blot du profil caracteristique de la proteine prion vMCJ. Apres automatisation de la phase de capture (automate Tecan Evo 100), la PMCA a ete optimisee en format microplaque et une technique ELISA de detection du prion a ete developpee en remplacement du Western-blot. Resultats Apres integration des 3 etapes, le test de 2 e generation a ete valide sur 72 echantillons controles avec une sensibilite identique au test manuel et le maintien d’une specificite de 100 %. Conclusions et perspectives L’utilisation du test automatise permet d’analyser 86 echantillons simultanement au lieu de 24 manuellement et permet donc d’augmenter la cadence d’un facteur 3,5. Ce test de 2 e generation pourra permettre de realiser des etudes de prevalence dans les populations a risque comme celle des polytransfuses.

Transfusion is a mixed discipline which includes the production of blood components, applied biology aiming in particular at establishing the highest compatibility for immunological characteristics between blood components to be delivered to patients and recipients, and, finally translational medicine to evaluate the effectiveness of the transfused products and to proactively avoid hazards, at least those that are preventible and can be anticipated. The whole chain takes place with the concern of continuous improvement of quality and safety. These two principles (quality/safety) have been and still are concerns of constant progress applicable to all the transfusion chain steps; they benefit from programs of Research and Development (R&D). Fundamental research, basic but also applied and clinical research, accompanies, in a constantly joint manner, scientific and medical progresses by providing new solutions to dampen the current problems and to prepare the future.

International audience

Several approaches can be used for the electrochemical detection of bacterial contamination. Their performance can be assessed by the ability to detect bacteria at very low concentrations within a short-time response. We have already demonstrated that a conductometric biosensor based on interdigitated thin-film electrodes is adapted to detect bacteria in clinical samples like serum and compatible with microfluidic fabrication. The type of interdigitated microelectrodes influences the performance of the biosensor. This was shown by the results obtained in this work. A magnetic-nanoparticles based immunosensor was designed using gold screen-printed electrodes. The immunosensor was able to specifically detect E. coli in the range of 1-103 CFU mL-1. The new transducer offered a larger active sensing surface with a lower cost and a robust material. Accuracy of the conductance value was enhanced by differential measurements. The immunosensor is compatible with a microfluidic system.

L’hemagglutination est la technique de reference pour la determination d’un phenotype erythrocytaire. Neanmoins, cette technique serologique presente des limites, et plus particulierement dans certaines situations ou le phenotypage erythrocytaire des patients ne peut etre realise. Dans ce contexte, la determination d’un genotype par biologie moleculaire permet d’obtenir un phenotype deduit pour le patient et ainsi de prevenir les risques d’allo-immunisation anti-erythrocytaire ou de limiter le risque d’incident transfusionnel. Pour repondre a cette problematique, nous avons developpe un test moleculaire rapide et innovant permettant de deduire visuellement le phenotype etendu dans le cadre de la prise en charge transfusionnelle du patient. Cette technologie repose sur la co-amplification par PCR multiplexe de plusieurs fragments d’ADN genomique directement a partir de sang total, et sur l’utilisation d’un support type membrane Lateral-Flow pour la lecture visuelle des resultats. Les amplicons biotinyles et les reactifs de revelation migrent par capillarite dans la membrane. L’apparition de spots colores, resultant de l’hybridation des amplicons biotinyles complexes avec les nanoparticules d’or et des sondes greffees sur la membrane, indique la presence des alleles correspondants. L’absence de coloration traduit l’absence des alleles concernes. Nous presentons les resultats de l’evaluation de cette technologie sur un panel d’echantillons de donneurs. Les resultats obtenus montrent que notre technologie brevetee, permet de deduire un phenotype erythrocytaire en une heure, pour six antigenes d’interet (JK1/JK2, FY1/FY2/FY2null et MNS3/MNS4).

Les symptomes cliniques communs aux infections arbovirales et leur co-circulation en zones endemiques rendent complexe leur diagnostic. Combiner les tests moleculaires et immunologiques sur une meme plateforme permettrait d’ameliorer la surveillance epidemiologique et la prise en charge des patients. Dans ce contexte, nous avons evalue une nouvelle approche analytique utilisant l’agglutination de nanoparticules superparamagnetiques (NPMag). Les genomes des virus Dengue (DENV) et ZIKA (ZIKV) sont amplifies par RT-PCR pan-flavivirus puis captures par des sondes polythiols greffees sur des NPMag. Ces sondes ont ete validees par ELOSA (test moleculaire en format microplaque). Le test immunologique utilise des NPMag fonctionalisees par des proteines virales recombinantes NS1 pour detecter les anticorps anti-DENV ou anti-ZIKV. Les agregats sont formes durant plusieurs cycles d’aimantation/relaxation puis detectes par une mesure d’absorbance. Une sensibilite analytique de 10–102 TCID50/mL est obtenue sur les gammes de reference (CNR Arboviroses). Sur 16 plasmas humains ZIKV positifs en RT-PCR (Ct

Le variant de la maladie de Creutzfeldt-Jakob (vMCJ) est une maladie a prion humaine rare dont l’issue est toujours fatale. Bien que tous les cas cliniques de vMCJ evalues pour le gene de la proteine prion, PRNP, aient ete homozygotes methionine au codon 129 depuis 20 ans, le premier patient heterozygote Methionine/Valine (MV) atteint de vMCJ a ete decrit en 2016 au Royaume-Uni. Ce patient repondait aux criteres diagnostiques de la MCJ sporadique probable (sMCJ). Ce nouveau cas souligne la possibilite d’une deuxieme vague de vMCJ et confirme la necessite du maintien de la surveillance. Objectif Developper un test pre-mortem visant a distinguer les patients vMCJ MV des sMCJ. Methode L’amplification cyclique des proteines par PMCA a deja montre de hautes performances dans la detection des prions vMCJ dans le sang notamment par l’identification du portage asymptomatique chez deux donneurs de sang qui ont developpe ulterieurement un vMCJ (Bougard et al., Sci Transl Med 2016). Cette technologie ultrasensible a ete adaptee a la detection specifique du vMCJ dans le liquide cephalorachidien (LCR) y compris chez le nouveau patient MV. Resultats Parmi les 98 LCR analyses en aveugle, nous avons identifie 40 des 41 echantillons de vMCJ. Ce test permet la discrimination du cas vMCJ heterozygote MV des 12 patients MV atteint de sMCJ. Il a egalement montre une specificite analytique de 100 % : aucun des 57 LCR provenant de patients atteints de sMCJ, de MCJ genetique, de maladie d’Alzheimer ou d’autres maladies neurologiques n’a donne de reaction positive. Conclusion Le test permet de realiser un diagnostic des patients vMCJ y compris chez les personnes heterozygotes MV avant leur deces.

Background Human neutrophil peptides (HNPs) 1 to 3 are the major antimicrobial peptides of the azurophilic granules of neutrophils. They represent an important arm of the innate immune system. Their production by chemical synthesis and recombinant technologies is expensive and limited by technical constraints due to their composition and the presence of three disulfide bonds. Study Design and Methods We have developed an original approach based on the purification of the natural human defensins HNPs 1 to 3 from neutrophils trapped on leukoreduction filters used in blood processing. The purification of HNPs 1 to 3 from these filters is performed in two steps: extraction of HNPs 1 to 3 retained in the filters followed by their immunoprecipitation. Studies were performed to determine the stability of defensins in the filters stored at room temperature. The activity of HNPs 1 to 3 obtained by our rapid protocol was validated by determining minimal inhibitory concentrations (MICs) against six reference bacterial strains and 12 clinical isolates. Results The human defensins HNPs 1 to 3 extracted from leukoreduction filters displayed high antimicrobial activity against tested strains, with MIC values between 0.12 and 1 μg/mL. Kinetics assays showed the appearance of activity 15 minutes after peptide addition. Moreover, we found that the HNPs 1 to 3 purified from leukoreduction filters that had been stored for 45 days at room temperature remained active. Conclusion Leukoreduction filters provide a rich and safe source of active human defensins HNPs 1 to 3. Moreover, the stability of the peptides in filters stored at room temperature allows envisaging a large-scale development of the process.

International audience

Recently, the flavivirus Zika virus (ZIKV) has rapidly spread in the Americas and the Caribbean islands. While a large proportion of infected persons are subjected to mild or asymptomatic disease, neurological disorders such as Guillain-Barre syndrome and microcephaly have been linked to ZIKV

Zika virus, discovered in 1947, is particularly publicized because of its involvement in a major epidemic that began in 2015 and which epicenter is located in Latin America, mainly in Brazil. In the majority of cases (70-80 %) the infection is asymptomatic, however in some patients, moderate fever, skin rash, conjunctivitis and myalgia may occur. More alarming, neurological complications are reported, in particular cases of microcephaly probably resulting from the infection of women in the first or second trimester of pregnancy. Moreover, Guillain-Barré syndromes have also been identified in patients whose infection was confirmed. The extent of the current outbreak reveals the very primitive state of knowledge about the pathophysiology of this virus. Thus, a global effort is being undertaken in order to quickly characterize the molecular interaction of the virus with human cells, but also to develop specific diagnostic assays and vaccinal approaches.

Zika virus, discovered in 1947, is particularly publicized because of its involvement in a major epidemic that began in 2015 and which epicenter is located in Latin America, mainly in Brazil. In the majority of cases (70-80 %) the infection is asymptomatic, however in some patients, moderate fever, skin rash, conjunctivitis and myalgia may occur. More alarming, neurological complications are reported, in particular cases of microcephaly probably resulting from the infection of women in the first or second trimester of pregnancy. Moreover, Guillain-Barre syndromes have also been identified in patients whose infection was confirmed. The extent of the current outbreak reveals the very primitive state of knowledge about the pathophysiology of this virus. Thus, a global effort is being undertaken in order to quickly characterize the molecular interaction of the virus with human cells, but also to develop specific diagnostic assays and vaccinal approaches.

Transfusion safety relating to blood-transmissible agents is a major public health concern, particularly when faced with the continuing emergence of new infectious agents. These include new viruses appearing alongside other known reemerging viruses (West Nile virus, Chikungunya) as well as new strains of bacteria and parasites (Plasmodium falciparum, Trypanosoma cruzi) and finally pathologic prion protein (variant Creutzfeldt-Jakob disease). Genomic mutations of known viruses (hepatitis B virus, hepatitis C virus, human immunodeficiency virus) can also be at the origin of variants susceptible to escaping detection by diagnostic tests. New technologies that would allow the simultaneous detection of several blood-transmissible agents are now needed for the development and improvement of screening strategies. DNA microarrays have been developed for use in immunohematology laboratories for blood group genotyping. Their application in the detection of infectious agents, however, has been hindered by additional technological hurdles. For instance, the variability among and within genomes of interest complicate target amplification and multiplex analysis. Advances in biosensor technologies based on alternative detection strategies have offered new perspectives on pathogen detection; however, whether they are adaptable to diagnostic applications testing biologic fluids is under debate. Elsewhere, current nanotechnologies now offer new tools to improve the sample preparation, target capture, and detection steps. Second-generation devices combining micro- and nanotechnologies have brought us one step closer to the potential development of innovative and multiplexed approaches applicable to the screening of blood for transmissible agents.

The development of multiplex and flexible tests allowing the simultaneous analysis of pathogens presenting a transfusional risk is a real challenge. Current miniaturized platforms have been particularly marked by microarrays. These microsystems allow the optical detection of hundreds of individual targets simultaneously. However, they suffer from a low sensitivity and their combination with a preliminary target amplification step such as PCR is necessary. The variable level of expression of the infectious genomes of interest and their large diversity complicate multiplex amplification. Finally simultaneous analysis of multiple blood-transmitted agents poses numerous difficulties in diagnosis that remain unresolved by currently available technologies. Until recently, scientific and technological advances for pathogen detection have focused on target amplification and optical detection steps. Today, sample preparation is recognized as a critical area to improve. Nanotechnologies can reach the single-cell or molecular scale and consequently overcome several current technological obstacles. They offer new technological tools for improving sample preparation but also for avoiding target amplification and the current fluorescent labeling. The combination of nano-objects and nano-systems in current technologies offers new possibilities for potential applications in the detection of infectious agents.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from the consumption of meat products contaminated by the agent causing bovine spongiform encephalopathy. Evidence supporting the presence of a population of silent carriers that can potentially transmit the disease through blood transfusion is increasing. The development of a blood-screening assay for both symptomatic vCJD patients and asymptomatic carriers is urgently required. We show that a diagnostic assay combining plasminogen-bead capture and protein misfolding cyclic amplification (PMCA) technologies consistently detected minute amounts of abnormal prion protein from French and British vCJD cases in the required femtomolar range. This assay allowed the blinded identification of 18 patients with clinical vCJD among 256 plasma samples from the two most affected countries, with 100% sensitivity [95% confidence interval (CI), 81.5 to 100%], 99.2% analytical specificity (95% CI, 95.9 to 100%), and 100% diagnostic specificity (95% CI, 96.5 to 100%). This assay also allowed the detection of silent carriage of prions 1.3 and 2.6 years before the clinical onset in two blood donors who later developed vCJD. These data provide a key step toward the validation of this PMCA technology as a blood-based diagnostic test for vCJD and support its potential for detecting presymptomatic patients, a prerequisite for limiting the risk of vCJD transmission through blood transfusion.

International audience

GB virus-C alias "hepatitis" virus G was discovered in 1995 as a putative causative virus of non A-E hepatitis. It is a very common virus found in 1 to 5% of eligible blood donors in developed countries. Numerous studies over seven years led to the exclusion of its role as a significant etiological agent of hepatitis. Its in vivo replication site is still unknown. Its direct involvement in the induction of significant hepatic or extra-hepatic diseases could not be demonstrated. However, coinfections with other viruses may contribute to changes in the evolution of both liver disease (negatively) and HIV/AIDS (favourably). Today, no country has decided to screen GBV-C in blood donors. However, more studies are necessary before the absence of influence of GBV-C infection on human health in the context of other viral infections could be confirmed definitely. This article is a review of the literature on a possible involvement of GBV-C in pathologies whether associated or not to other infections.

La contamination bacterienne des produits sanguins labiles represente actuellement le premier risque infectieux transfusionnel. Impliques dans plus de 80 % des incidents, les concentres plaquettaires sont les PSL les plus a risque. La faible concentration bacterienne au moment du don (0,03–0,3 CFU/mL) et les differentes cinetiques de croissance observees selon les souches impliquees, Gram − ou Gram + rendent complexe le developpement d’un test de diagnostic. Ainsi, un test performant de detection des bacteries dans les PSL doit non seulement etre generique afin de detecter l’ensemble des souches, mais aussi hautement sensible pour detecter precocement de faibles concentrations. De nombreux pays ont mis en place des approches preventives basees sur des methodes de culture sensibles, mais longues ou sur des tests plus rapides, mais moins sensibles. D’autres pays se sont orientes sur les methodes de reduction de charge, efficaces mais tres couteuses. Le developpement d’un test combinant a la fois les performances de sensibilite et de rapidite est un enjeu important afin de delivrer le plus tot possible des PSL qualifies. Dans ce contexte, nous avons developpe avec un partenariat industriel une nouvelle approche economique combinant une etape de culture et un test immunologique. Des microparticules magnetiques fonctionnalisees par des anticorps generiques selectionnes de maniere innovante permettent d’atteindre les criteres de performances requis. Ces outils biologiques sont flexibles, adaptes a une detection optique ou electrochimique, en format microplaque automatisable pour du moyen debit ou en format capteurs pour des tests portables.

Transfusion is a mixed discipline which includes the production of blood components, applied biology aiming in particular at establishing the highest compatibility for immunological characteristics between blood components to be delivered to patients and recipients, and, finally translational medicine to evaluate the effectiveness of the transfused products and to proactively avoid hazards, at least those that are preventible and can be anticipated. The whole chain takes place with the concern of continuous improvement of quality and safety. These two principles (quality/safety) have been and still are concerns of constant progress applicable to all the transfusion chain steps; they benefit from programs of Research and Development (RD). Fundamental research, basic but also applied and clinical research, accompanies, in a constantly joint manner, scientific and medical progresses by providing new solutions to dampen the current problems and to prepare the future.

La prevention du risque infectieux represente un enjeu majeur de securite transfusionnelle face a l’emergence continue d’agents transmissibles par le sang. Les besoins de multiplexage (visant a depister simultanement de nombreux virus connus ou emergents), de flexibilite, et, de reduction des couts d’analyse, conduisent a poursuivre le developpement d’approches diagnostiques innovantes. Dans ce contexte, nous avons mis au point de nouveaux outils moleculaires permettant, dans un format simplifie sur support microplaque, l’analyse de genomes viraux. La capture d’acides nucleiques amplifies (cibles) a ete fortement optimisee par le recours a une chimie innovante de synthese d’oligonucleotides polythiols (sondes) [1] , [2] . L’hybridation sondes/cibles est mesuree par fluorescence en temps resolu (DELFIA). Une premiere evaluation a ete realisee sur les panels de reference (WHO, NIBSC) et a montre des sensibilites analytiques atteignant 100 IU/mL, 10 IU/mL and 50 IU/mL, respectivement pour les virus HCV, HBV and HIV. Cette approche a ete ensuite etendue au genotypage HCV [3] en couplant une amplification generique des genomes dans la region NS5B (401 nt) et leur capture via des sondes polythiols specifiques 1a/1b, 2a/2b/2c, 3a et 4a/4d. L’analyse d’un panel de plasma VHC (+) code ( n = 100) montre une correlation de 100 % entre notre technique et la methode de reference par sequencage. Des developpements sont en cours pour la detection de virus emergents (West Nile, Dengue, Chikungunya). L’utilisation de sondes polythiols pourra etre exploitee pour le developpement de plateformes automatisees generiques adaptees au depistage et au genotypage multiplexe de virus.

International audience; The aim of this study was to develop versatile diagnostic tools based on the use of innovative polythiolated probes for the detection of multiple viruses. This approach is compatible with optical enzyme-linked oligosorbent assay (ELOSA) or electrochemical (biosensors) detection methods. The application targeted here concerns the rapid genotyping of Hepatitis C virus (HCV). HCV genotyping is one of the predictive parameters currently used to define the antiviral treatment strategy and is based on the sequencing of the viral NS5b region. Generic and specific NS5b amplicons were produced by real-time polymease chain reaction (RT-PCR) on HCV(+) human plasma. Original NS5b probes were designed for genotypes 1a/1b, 2a/2b/2c, 3a, and 4a/4d. Robust polythiolated probes were anchored with good efficacy on maleimide-activated microplates (MAM) and gold electrodes. Their grafting on MAM greatly increased the sensitivity of the ELOSA test which was able to detect HCV amplicons with good sensitivity (10 nM) and specificity. Moreover, the direct and real-time electrochemical detection by differential pulse voltammetry enabled a detection limit of 10 fM to be reached with good reproducibility. These innovative polythiolated probes have allowed us to envisage developing flexible, highly sensitive, and easy-to-handle platforms dedicated to the rapid screening and genotyping of a wide range of viral agents.

International audience; : Blood safety is a global health goal. In developed countries, bacterial contamination of platelet concentrates is the highest infectious risk in transfusion despite the current preventive strategies. We aimed to develop a conductometric biosensor for the generic, rapid and sensitive detection of Gram-negative bacteria. Our strategy is based on immunosensors: addressable magnetic nanoparticles coupled with anti-LPS antibodies were used for the generic capture of Gram-negative bacteria. Bacterial capture was characterized by impedancemetric and microscopic measurements. The results obtained with conductometric measurements allowed real-time, sensitive detection of Escherichia coli or Serratia marcescens cultures from 1 to 10(3)CFUmL(-1). The ability of the immunosensor to detect Gram negative bacteria was also tested on clinically relevant strains. The conductometric immunosensor allowed the direct detection of 10-10(3)CFUmL(-1) of Pseudomonas aeruginosa and Acinetobacter baumannii strains that were undetectable using standard immunoblot methods. Results showed that the conductometric response was not inhibited in 1% serum.

International audience; Understanding the virus-host interactions that lead to approximately 20% of patients with acute Hepatitis C Virus (HCV) infection to viral clearance is probably a key towards the development of more effective treatment and prevention strategies. Acute hepatitis C infection is usually asymptomatic and therefore rarely diagnosed. Nevertheless, HCV nucleic acid testing carried out on all blood donations detects donors who have resolved their HCV infection after seroconver-sion. Here we have used SELDI-TOF-MS technology to compare, at a proteomic level, plasma samples respectively from donors with HCV clearance, from donors with chronic HCV infection and from unexposed healthy donors (n = 15 per group). A candidate marker of about 9.4 kDa was detected as differentially expressed in the three groups. After purification we identified by nanoLC-Q-TOF-MS/MS this candidate marker as Apolipoprotein C-III (ApoC-III). The identification was confirmed by western blot analysis. Levels of ApoC-III were then determined in the 45 plasma samples by immunoturbidimetric assay. ApoC-III was found to be higher in donors who had resolved their HCV infection than in donors with chronic infection, results which were consistent with SELDI-TOF-MS data. ApoC-III is the first reported candidate biomarker in plasma associated with the spontaneous resolution of HCV infection.

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.

International audience; The low but known risk of bacterial contamination has emerged as the greatest residual threat of transfusion-transmitted diseases. Label-free detection of a bacterial model, Escherichia coli, is performed using nonfaradic electrochemical impedance spectroscopy (EIS). Biotinylated polyclonal anti-E. coli is linked to a mixed self-assembled monolayer (SAM) on a gold electrode through a strong biotin-neutravidin interaction. The binding of one antibody molecule for 3.6 neutravidin molecules is determined using the surface plasmon resonance (SPR). The detection limit of E. coli found by SPR is 10(7) cfu/mL. After modeling the impedance Nyquist plot of E. coli/anti-E. coli/mixed SAM/gold electrode for increasing concentrations of E. coli (whole bacteria or lysed bacteria), the main parameter that is modified is the polarization resistance R-P. A sigmoid variation of R-P is observed when the log concentration of bacteria (whole or lysed) increases. A concentration of 10 cfu/mL whole bacteria is detected by EIS measurements while 10(3) cfu/mL is detected for lysed E. coli.

International audience; BACKGROUND/AIMS: The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point. METHODS: Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively. Infection was performed in the absence or presence of LDL, HDL, recombinant soluble LDLR peptides encompassing full-length (r-shLDLR4-292) or truncated (r-shLDLR4-166) LDL-binding domain, monoclonal antibodies against r-shLDLR4-292, squalestatin or 25-hydroxycholesterol. Intracellular amounts of replicative and genomic HCV RNA strands used as end point of infection were assessed by RT-PCR. RESULTS: r-shLDLR4-292, antibodies against r-shLDLR4-292 and LDL inhibited viral RNA accumulation, irrespective of genotype, viral load or liver donor. Inhibition was greatest when r-shLDLR4-292 was present at the time of inoculation and gradually decreased as the delay between inoculation and r-shLDLR4-292 treatment increased. In hepatocytes pre-treated with squalestatin or 25-hydroxycholesterol before infection, viral RNA accumulation increased or decreased in parallel with LDLR mRNA expression and LDL entry. CONCLUSIONS: LDLR is involved at an early stage in infection of normal human hepatocytes by serum-derived HCV virions.