Joliette Coste

In vitro infection of adult normal human hepatocytes in primary culture by hepatitis C virus

In vitro infection of adult normal human hepatocytes in primary culture has been performed for investigating the replication cycle of hepatitis C virus (HCV) in differentiated cells. Hepatocytes were prepared from liver tissue resected from donors who tested negative for HCV, and inoculation was performed 3 days after plating with 33 HCV serum samples of different virus load and genotype. The presence of intracellular HCV RNA, detected by a strand-specific rTth RT-PCR assay, was used as evidence of infection. A kinetics analysis of HCV replication revealed that intracellular negative-strand RNA appeared at day 1 post-infection with a maximum level at days 3 and 5, followed by a decrease until day 14. At day 5, we estimated that the copy level of viral RNA was amplified at least 15-fold in infected cells. The level of intracellular HCV RNA in response to different serum samples was reproducible from one hepatocyte culture to another, suggesting that there is no inter-individual variability in the susceptibility of hepatocytes to HCV infection. These findings indicate that adult human hepatocytes in primary culture retain their susceptibility to in vitro HCV infection and support HCV RNA replication. This model should represent a valuable tool for the study of initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Serum-Derived Hepatitis C Virus Infection of Primary Human Hepatocytes Is Tetraspanin CD81 Dependent

ABSTRACT 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.

Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

ABSTRACT Chronic hepatitis C is a common cause of liver disease, the complications of which include cirrhosis and hepatocellular carcinoma. Treatment of chronic hepatitis C is based on the use of alpha interferon (IFN-α). Recently, indirect evidence based on mathematical modeling of hepatitis C virus (HCV) dynamics during human IFN-α therapy suggested that the major initial effect of IFN-α is to block HCV virion production or release. Here, we used primary cultures of healthy, uninfected human hepatocytes to show that: (i) healthy human hepatocytes can be infected in vitro and support HCV genome replication, (ii) hepatocyte treatment with IFN-α results in expression of IFN-α-induced genes, and (iii) IFN-α inhibits HCV replication in infected human hepatocytes. These results show that IFN-α acts primarily through its nonspecific antiviral effects and suggest that primary cultures of human hepatocytes may provide a good model to study intrinsic HCV resistance to IFN-α.

Comparative evaluation of three assays for the quantitation of human immunodeficiency virus type 1 RNA in plasma

Reverse transcriptase‐coupled polymerase chain reaction (Amplicor HIV‐1 Monitor), the branched DNA (bDNA) method (Quantiplex HIV‐1 RNA) and the nucleic acid sequence‐based assay (NASBA HIV‐1 RNA QT) were comparatively evaluated for the quantitation of human immunodeficiency virus type 1 (HIV‐1) RNA in plasma. Among 60 plasma specimens from HIV‐1 infected patients, HIV‐1 RNA was detected in 56 by Amplicor (sensitivity, 93.3%), in 41 by bDNA (sensitivity, 68.3%), and in 60 by NASBA (sensitivity, 100%). HIV‐1 RNA was not detected by any of these methods in 34/34 plasma specimens from HIV‐1‐seronegative blood donors (specificity, 100%). The HIV‐1 RNA levels as determined by the different methods were correlated significantly. The frequency of concordant results (log difference <0.50) was 80.4% between Amplicor and NASBA, 77.5% between Amplicor and bDNA, and 58.6% between bDNA and NASBA. After initiation of antiviral therapy, HIV‐1 RNA level variations observed with the three methods were similar. HIV‐1 RNA levels were inversely correlated with the CD4+ T cell counts, whereas no correlation was found with HIV‐1 p24‐antigen levels. When the methods were evaluated for reproducibility, coefficients of variation ranged from 11% to 40% for Amplicor, from 6% to 35% for bDNA, and from 13% to 62% for NASBA. Quantitation of HIV‐1 RNA in culture supernatants from HIV‐1 subtype A to H strains showed that bDNA can be used to quantitate RNA from all HIV‐1 subtypes, whereas Amplicor failed to detect RNA from subtype A strains and NASBA subtype G strains.

Evaluation of the enhanced bacterial detection system for screening of contaminated platelets

BACKGROUND:  The Pall third‐generation enhanced bacterial detection system (eBDS) was recently approved for detection of bacterial contamination in leukoreduced platelets (PLTs). The method is based on the measurement of the oxygen content as a marker for bacteria. eBDS incorporates major modifications including removal of the sample‐set filter, modification of the culture medium, and incubation with agitation of the sample pouch.

Detection of prions in the plasma of presymptomatic and symptomatic patients with variant Creutzfeldt-Jakob disease

A sensitive blood diagnostic assay can identify patients with variant Creutzfeldt-Jakob disease before symptoms appear and during the clinical phase. A new blood test for prion diseases Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease that can be transmitted from person to person through medical procedures. The development of a diagnostic blood test is an urgent priority. Bougard et al. describe a sensitive and specific blood test based on a prion capture step and an amplification method. This test for vCJD was very accurate and worked not only for blood samples from patients suffering from vCJD but also for samples taken from two individuals 1.3 and 2.6 years before they developed clinical symptoms. This blood test has important implications for transfusion medicine and public health. 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.

Microconductometric immunosensor for label-free and sensitive detection of Gram-negative bacteria

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) CFU mL(-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) CFU mL(-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.

Detection of blood-transmissible agents: can screening be miniaturized?

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.

Plasminogen-Based Capture Combined with Amplification Technology for the Detection of PrPTSE in the Pre-Clinical Phase of Infection

Background Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrPTSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrPTSE concentrations in the femtomolar range. Methodology/Principal Findings We have developed a three-step assay that firstly captures PrPTSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrPTSE detection by western blot. We achieved a PrPTSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrPTSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrPTSE in human plasma spiked with a 10−8 dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrPTSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples. Conclusion/Significance We have developed a sensitive and specific amplification assay allowing the detection of PrPTSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrPTSE in blood of patients displaying positivity in large scale screening tests.

Feasibility study of a screening assay that identifies the abnormal prion protein PrPTSE in plasma: initial results with 20,000 samples.

BACKGROUND: It is likely that transmission of variant Creutzfeldt‐Jakob disease (vCJD) occurs by transfusion and that the candidate infectious agent (PrPTSE) is present in small concentrations in the blood of infected donors in the asymptomatic phase of the disease. A new blood screening assay has been developed to detect PrPTSE in citrated plasma samples.