Multicenter clinical evaluation of the new 3rd generation assay for detection of antibodies against hepatitis C virus on the VIDAS(®) system.

BACKGROUND: Detection of antibodies (anti-HCV) against hepatitis C virus (HCV) is indispensable for screening and diagnosis of viral hepatitis and for the viral safety of blood, tissue or organ donations. It gains additional importance by the new HCV drugs which improve the therapeutic possibilities dramatically. OBJECTIVE: To evaluate the performance of a newly developed immune assay for anti-HCV based on the well-established VIDAS platform. STUDY DESIGN: The assay was evaluated with samples from anti-HCV negative blood donors and from patients with or without HCV markers in six centres in France, Spain and Egypt. The status of the samples was determined by using CE-marked immune assays (Architect, AxSym, Prism, Vitros), two immunoblots (RIBA, Inno-Lia) and/or HCV RNA results. RESULTS: Specificity was 99.67% in 10,320 French blood donors without anti-HCV, 99.5% in 200 anti-HCV negative hospitalized European patients and 99.0% in 198 negative patients from Egypt. Sensitivity was 99.7% in 1054 patients pretested positive by other assays; 345 patients with known genotype had genotype 1-6; 61 patients were co-infected with HIV. VIDAS was reactive in 78% of 91 patients with uncertain or very weak anti-HCV. It became on average positive at day 37 with seroconversion panels. CONCLUSIONS: This multicentric, international study with >12,000 samples show that the new VIDAS anti-HCV assay is very suitable for screening and confirmation of HCV infection. Sensitivity, specificity and recognition of seroconversion compare favorably with well-established CE-marked tests and help to clarify discrepant results obtained with other assays.

[Technological evolutions in blood donation screening and their impact on the residual risk]. / Évolutions technologiques en qualification biologique du don et leur impact sur le risque résiduel transfusionnel.

During the two last decades, the risk of viral transfusion transmission for some infectious agents (HIV, HCV, HBV and HTLV) has been markedly reduced by improved donor screening, improvements of serological assays and the implementation of minipool nucleic acid testing for HIV-1 and HCV viruses. However, implementation during the year 2010 of nucleic acid testing for the detection of HIV RNA, HCV RNA and HBV DNA in a single triplex assay may provide additional safety, especially after acute infection during the window period. New Procleix(®) Tigris(®) technology (Novartis) allow the French blood screening laboratories to answer their actual requirements in terms of security and throughput and to implement nucleic acid testing in case of emergent risks requiring a direct detection of viruses. Furthermore, Tigris(®) is a fully automated system with high level of security during the analytical process, reducing the number of invalid or non-available results observed with the previous semi-automated technologies. Moreover, renewal of material by fully automated and secure systems, especially for the critical step of sample pipeting, may provide additional safety in blood screening laboratories.

Multicentric evaluation of the DiaMed enzyme-linked immunosorbent assay malaria antibody test for screening of blood donors for malaria.

BACKGROUND: The risk of malaria transmission by blood transfusion is critical due to extensive travel from endemic areas to non-endemic areas. An enzyme-linked immunosorbent assay (ELISA) malaria antibody test has been developed that is claimed to perform better than the immunofluorescence assay test (IFAT). The assay contains antigens to both Plasmodium falciparum and Plasmodium vivax. A multicentre study was performed to evaluate the appropriateness of replacing the IFAT by the new ELISA test. MATERIAL AND METHODS: Nine French blood banks participated in this multicentre study. Two panels of samples were evaluated. The first included 4163 samples from healthy donors and was used to calculate clinical specificity of the assay. The second involved 10,995 samples, either collected retrospectively or prospectively from malaria-risk donors , was used to assess the comparative performance of the ELISA and IFAT. Discordant samples were further tested using an in-house IFAT and also tested for presence of Plasmodium DNA by polymerase chain reaction. RESULTS: The ELISA showed a clinical specificity of 99.02%. In the malaria-risk blood donors groups, the retrospective group showed a concordance rate of 92.6% (k = 0.90), while the prospective group showed a concordance rate of 97% (k = 0.46). After confirming the discordant sample results by an in-house IFAT, the k index increased to 0.81. None of the discordant samples was shown to contain Plasmodium DNA. CONCLUSION: The performance of the ELISA test in this study has confirmed its potential as a new screening test for use in blood banks, as an alternative to the IFAT in prevention of transfusion-transmitted malaria in non-endemic countries.

[Application of molecular biology to blood transfusion safety: the nucleic acid testing]. / Application de la biologie moléculaire à la sécurité virale transfusionnelle: le dépistage génomique viral.

Nucleic Acid Testing (NAT) for HIV-1 and HCV has been introduced in France and became mandatory for all homologous blood donations since July 1st 2001 in addition to serology screening. Previously, a feasibility study led to the selection of 2 technologies : a TMA based assay (Chiron) uses the Procleix HIV-/HCV assay on pools of 8 samples and a PCR based assay from BioMérieux-Roche which is a combination of an RNA extraction system (NucliSens, BioMérieux) with a fully automated system for nucleic acid amplification and detection (Cobas Amplicor, Roche). This system uses the Cobas Ampliscreen HCV test v.2.0 and the The Cobas Ampliscreen HIV test v1.5, on 24 sample pools. Pooling was required because single-donation testing is not yet feasible, as a result of the limitations in automation available for all current NAT technologies. The two technologies were easily implemented and showed nearly the same detection limit for HCV RNA and HIV-1 RNA. During a one-year period, from July 1st 2001 to June 30, 2002, out of the 2.5 million donations tested, the NAT yield resulted in one HIV-1 RNA positive-antibody negative donation and one HCV RNA positive-antibody negative donation. Two HIV NAT positive-antibody negative donations were missed by minipool NAT because a very low viral load. Moreover, the NAT implementation did not impact on blood component availability, including platelet concentrates.

Hepatitis C virus: routes of infection and genotypes in a cohort of anti-HCV-positive French blood donors.

BACKGROUND: We evaluated and analysed risk factors of HCV-infected blood donors according to HCV genotypes in order to improve the transfusion policy and safety of blood supply. MATERIALS AND METHODS: HCV-RNA was analysed in sera from 518 anti-HCV-positive blood donors, who were invited to medical consultation and interview as to risk factors by means of an extensive questionnaire. HCV genotyping was done on all samples positive for HCV-RNA. RESULTS: Of the 518 sera, 399 (77%) were HCV-RNA positive, and 394 of 399 HCV genotypes were identified. Major genotypes were 1b (34.3%), 3a (24%), 1a (19.5%) and 2 (11.4%). Of the donors, 289 (55.8%) were interviewed regarding their risk behaviour: 27% were former intravenous drug users (IVDUs), 26% had been transfused, 8% had a history of invasive diagnostic procedures, and 13% a history of surgery. Among the 224 interviewed donors, genotypes 1a and 3a were mainly associated with IVDU (51 and 45% respectively) and genotype 1b, with transfusion and nosocomial infections (40 and 25%, respectively). CONCLUSION: In this population of anti-HCV-positive blood donors, nosocomial infection may be a route of HCV spread, but the main risk factor remains IVDU, particularly in young men. The transfusion policy will improve if predonation interviews of such young men are done with a specific and sensitive questionnaire.

HCV RNA in blood donors with isolated reactivities by third-generation RIBA.

BACKGROUND: The objective of this collaborative study was to learn the proportion of HCV RNA-positive samples obtained from a population of donors with isolated anti-HCV reactivities by third-generation RIBA (RIBA-3) (indeterminate results). STUDY DESIGN AND METHODS: During a 2-year period, 11 blood transfusion centers kept all samples with indeterminate RIBA-3 results to test them by PCR, using both local and commercial techniques. RESULTS: Of the 758 RIBA-3 indeterminate samples, 10 (1.3%) were positive for HCV RNA: 3. 3 percent (6/180) and 1.3 percent (4/317) of samples with anti-core or anti-NS3 reactivity, respectively, and none of the 52 and 209 samples with anti-NS4 or anti-NS5 reactivity, respectively. HCV RNA-positive donors with anti-core reactivity were infected with different subtypes (1 with HCV subtype 1b, 1 with 2, 1 with 2a/2c, 2 with 3a, and 1 with 5a), and a follow-up indicated a chronic-carrier state in two of the six donors. Acute hepatitis was diagnosed in three of the four donors with anti-NS3 reactivity alone. Two of these three were IV drug users and were infected with subtype 1a. CONCLUSION: HCV RNA-positive donors with indeterminate results in RIBA-3 are extremely rare, but they do exist. They were observed only when either anti-core or anti-NS3 was present. With such a RIBA-3 profile, PCR testing remains necessary to reveal an eventual acute or chronic HCV infection.

HCV-Infection in blood donors: association between anti-HCV core IgM antibodies and serum HCV RNA.

Among 47 blood donors tested positive with HCV EIA 2.0 Abbott, 27 (57.4%) also reacted with four ¿third-generation' EIAs. The presence of anti-HCV antibodies was confirmed with 3 different immunoblot assays in 16 of 27 sera (34.0%) while 10 samples (21.3%) had indeterminate profile with antibodies usually directed against structural core antigen. Anti-HCV core IgM response was found in 12 of 47 sera (25.5%) and HCV viremia detected by the polymerase chain reaction (PCR) procedure was observed in 15 samples (31.9%). A comparative study of the different markers confirmed a good correlation between a strong antibody response in EIAs and immunoblot assays and the presence of HCV RNA in the serum; only 2 immunoblot indeterminate samples were PCR positive. An association was observed between IgM antibodies against "core' epitopes and HCV RNA carriage: all IgM-positive sera were found positive by PCR. However, the direct detection of viral genome remains the best method for identifying HCV carriers in the blood donor population.

Different prevalence of precore mutants in five members of a hepatitis-B-virus-infected family: evidence for a precore variant type in an asymptomatic anti-HBs patient.

Chronic active hepatitis B (CAH-B), anti-HBe (+) has been associated with a hepatitis B virus variant carrying a stop codon at the distal pre-C region that prevents HBeAg synthesis. We analyzed the HBV DNA pre-C region in five members of a Turkish family. The mother presented an anti-HBe (+) CAH-B and the four children different hepatitis B virus serological and clinical profiles. The pre-C region was analyzed by cloning after DNA amplification in sera and peripheral blood mononuclear cells. A method for rapid screening of a large number of cloned polymerase chain reaction products was developed for the presence of the most frequent pre-C mutations (G to A substitution at nucleotide position 1896 and 1899). At least 60 independent clones were tested for each patient by selective oligonucleotide hybridization using non-mutated (M0), one (M1) and two (M2) point-mutated probes. Results were confirmed by sequencing. The mutation 1896 was present in 91% of DNA clones from the mother. The same mutation was also found in 85% of the clones in the youngest child (D), but in less than 10% of the clones from children A and C. Only the pre-C wild-type strain was observed in child B. X gene deletions (3 to 20 nt) were also present in some clones from the mother and children A, B and C. No significant difference between serum and peripheral blood mononuclear cells concerning the viral population was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative detection of hepatitis B virus DNA in serum using chemiluminescence: comparison with radioactive solution hybridization assay.

A quantitative, non-radioactive hybrid capture HBV DNA assay (Digene Diagnostics), which uses an efficient solution hybridization procedure coupled to a sensitive chemiluminescent signal amplification system, was compared with the quantitative, radioactive solution hybridization assay (Genostics, Abbott Laboratories), in hepatitis B virus carriers, particularly in those undergoing antiviral therapy. The qualitative reproducibility of the chemiluminescent method, tested on 30 sera, was acceptable, with a reproducibility rate of 93.3%. A comparison of this hybrid capture HBV DNA assay with the radioactive test on 113 sera obtained from 48 patients (39 HBsAg-positive patients) gave a sensitivity of 87.2%, a specificity of 100% and an agreement between the two tests of 89.4% (101 sera including 82 HBV DNA positive and 19 negative samples). Changes in HBV DNA levels measured by the two assays showed a good correlation with each other during interferon therapy. However, the hybrid capture values were higher than the radioactive assay values, with the ratio of the two values being variable in the same patient during the course of treatment. The Genostics assay therefore seems to be a more accurate procedure for evaluating changes in viral replication, particularly at high HBV DNA levels. However, the hybrid capture method is faster and has the advantage of being a non-radioactive procedure. This chemiluminescent assay is easy to perform as a routine diagnostic procedure and may be a useful alternative to the radioactive solution hybridization method.

Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates.

The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.

Advantage of PCR for detecting low amounts of HBV DNA in patients' sera.

Serum hepatitis B virus DNA (HBV DNA) is now the most important and reliable marker for monitoring viral replication. Quantitative detection of HBV DNA in serum is based on a commercial standardized solution hybridization assay (Genostics). In this work, we studied the sensitivity and specificity of this method, in comparison with the polymerase chain reaction (PCR) technique, for low-value HBV DNA serum samples. Fifty-four patients with or without HBV serological markers were divided into 4 groups according to their HBV DNA values. Genomic amplification was found to affect 2 conserved regions of the viral genome, the S and C regions. Samples with an HBV DNA concentration equal to or greater than 1.5 pg/ml were considered positive in the "Genostics" test. A total of 38% of patients considered negative in the quantitative assay (less than 1.5 pg/ml) were found to be positive for HBV DNA in serum after PCR. Only 26% of patients with an HBV DNA concentration of between 1.5 and 10 pg/ml in the Genostics test had PCR-detectable viral DNA in serum. Some 56% of patients with HBV DNA values between 10 and 20 pg/ml were found to be positive after amplification. All patients whose HBV DNA values were above 20 pg/ml had PCR-detectable viral DNA in serum. Our PCR results suggest that the positive limit level of the Genostics test has to be re-evaluated. Indeed, for low values of HBV DNA (under 20 pg/ml and especially under 10 pg/ml), it is not possible to conclude about the positivity from the quantitative assay, and results have to be estimated according to the clinical and serological status of the patients. Moreover, PCR can be falsely negative because of methodological problems. Nevertheless, this study confirms that PCR does enable detection of the viral genome in HBV-seronegative patients and in "old" and "cured" HBV-infection marker carriers.