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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

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.

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.

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.

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.

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; : 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; 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.

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.