COBAS AmpliScreen Research Articles

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.

Comparative yield of HCV RNA testing in blood donors screened by 2.0 versus 3.0 antibody assays

Two HCV antibody tests (EIA 2.0 [EIA2], Abbott; and the Version 3.0 ELISA [EIA3], Ortho) are currently licensed for screening of US blood donors. Testing of donors for HCV RNA allows comparison of the sensitivities of the two antibody-screening assays. All allogeneic blood donations at 13 US test sites were screened for HCV RNA by testing plasma minipools using an investigational assay (COBAS AmpliScreen HCV test, v2.0, Roche Molecular Systems). Some sites screened for HCV antibody by EIA2 and some used EIA3. The frequency of RNA-positive and antibody-negative (RNA-pos and Ab-neg) donations among donors screened by each antibody assay was compared. Antibody appearance was assessed in a donor follow-up study. A total of 5.51 x 10(6) donations were screened for HCV RNA. Of these, 2.27 million were screened for antibody by EIA2, and 3.24 million by EIA3. Twenty-three donations were HCV RNA-pos and Ab-neg. The frequency of RNA-pos and Ab-neg donations was higher among donations screened by EIA2 (1 in 134,000), compared to those screened by EIA3 (1 in 540,000) (p = 0.001). Of the 17 RNA-pos and Ab-neg donations identified by test sites that used EIA2, 14 were retested by EIA3 and 10 (71%) were reactive. Most RNA-pos and Ab-neg donors appear to be in the process of seroconversion. Donors that were initially EIA2-negative and EIA3-reactive showed a more prolonged pattern of seroconversion compared to those that were initially nonreactive by both antibody assays. Four donors were EIA2-negative, EIA3-reactive, and RIBA-indeterminate (c33c) for at least 90 days, 1 for more than 317 days. EIA3 would have detected the majority of RNA-positive donations missed by EIA2. Some RNA-positive donors are EIA2-negative and EIA3-reactive for a prolonged period of time.

Nucleic acid testing: Update and applications

Nucleic acid testing (NAT) holds the promise of closing the window of infectiousness for hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) in the general blood supply. Pioneering work by the source plasma industry with NAT for hepatitis A virus (HAV), hepatitis B virus (HBV), and parvovirus B19 suggests that, in the future, the risk of other viral infections may be reduced using similar technology. The European Commission decree that, by July 1999, all source plasma for fractionation should be NAT nonreactive for HCV at a sensitivity of 100 viral IU/mL, has driven the implementation of NAT in the United States. It is estimated that more than 95% of the US blood supply is currently tested by one of two investigational tests for HCV and HIV, and many institutions restrict the release of red blood cell (RBC) and plasma products prior to the release of NAT results. NAT implementation has been hampered by a lack of fully automated, low-cost technologies; the absence of Food and Drug Administration (FDA)-approved and validated clinical tests; and lagging turnaround times. Results from US investigational trials of the Procleix Transcription Mediated Amplification (TMA) HCV/HIV (Chiron Corp, Emeryville, CA) and the COBAS AmpliScreen (Roche Diagnostics, Indianapolis, IN) polymerase chain reaction (PCR) assays have begun to substantiate their value. While NAT assays will not replace serologic tests, they lay the groundwork for further reducing the already low risk of infection transmission through transfusion of blood components and their factor derivatives.

Performance characteristics of the COBAS AmpliScreen HIV-1 test, version 1.5, an assay designed for screening plasma mini-pools.

The COBAS AmpliScreen HIV-1 test, version 1.5 (v1.5) (Roche Molecular Systems), is designed for screening pools composed of samples from 24 individual units of blood or plasma. A specimen-processing procedure (Multiprep) simultaneously concentrates and extracts HIV-1, HCV, and HBV particles from plasma and incorporates an HIV-1 internal control (IC) RNA. Processed samples are amplified by RT-PCR using HIV-1-specific primers and detected by hybridization of the amplified products to HIV-1- and IC-specific oligonucleotide probes. Plasma samples containing known quantities of HIV-1 were used to evaluate analytical sensitivity and precision and to validate a pool testing algorithm. Analytical specificity was evaluated by adding various viruses and bacteria to HIV-1-negative plasma. Seroconversion panels were tested to estimate the window-period reduction achieved by RNA testing. The analytical sensitivity of the test (concentration that yields > or = 95% positive results in a set of replicate tests) was 25 copies of HIV-1 RNA per mL of pooled plasma. Representative strains from all HIV-1 group M subtypes were reproducibly detected (> 95% positive results) at concentrations of 20 to 200 viral particles per mL. The test did not cross-react with a set of 31 viral and 5 bacterial isolates, and it yielded negative results on a panel of 500 blood samples from HIV-1-seronegative donors. Plasma samples containing abnormally high levels of Hb, albumin, triglycerides, or bilirubin did not interfere with the test. HIV-1 RNA was detected 2 to 14 days before HIV-1 antibody and 0 to 28 days before p24 antigen. The test specifically detected pools containing a single positive unit with 2400 HIV-1 RNA copies per mL and correctly identified the positive unit. The COBAS AmpliScreen HIV-1 test, v1.5, has sufficient sensitivity to detect a single infected unit containing 600 copies of HIV-1 per mL in a pool with 23 uninfected units and should reduce the window period between infection and seroconversion by at least 2 to 14 days.

Performance Characteristics of the AmpliScreenTMHIV-1 Test, an Assay designed for Screening Plasma Mini-pools

This study evaluated the performance characteristics of the AmpliScreenTMHuman Immunodeficiency Virus-Type 1 (HIV-1) Test, Version 1·5, a test designed for screening pools composed of samples from individual units of blood or plasma. HIV-1, hepatitis C (HCV) and hepatitis B (HBV) virus particles were simultaneously extracted and concentrated from plasma by a multi-prep sample processing procedure. An HIV-1 Internal Control (IC) RNA was added to each sample to serve as an extraction and amplification control. Processed samples were amplified by RT-PCR using HIV-1-specific complementary primers and detected by hybridization of the amplified products to HIV-1- and IC-specific oligonucleotide probes.The analytical sensitivity of the test (concentration that yields ≥95% positive results in a set of replicate tests) was 25 copies of HIV-1 RNA per mL of pooled plasma. Representative strains from all HIV-1 group M subtypes were reproducibly detected (>95% positive results among 22 replicate tests) at concentrations of 30 to 75 viral particles per ml. The test exhibited excellent specificity; it did not cross-react with a set of 30 viral and five bacterial isolates and yielded negative results on a panel of 500 blood samples from HIV-1 seronegative donors. Samples containing abnormally high levels of haemoglobin, albumin, triglycerides or bilirubin in plasma samples did not interfere with the detection of HIV-1 RNA at a concentration of 100 copies of per ml. The test detected HIV-1 RNA 7–17 days prior to anti-HIV-1 antibody seroconversion for all 10 seroconversion panels tested. A fully automated COBAS AmpliScreenTMversion of this test is being validated. COBAS AmpliScreen tests for HCV and HBV also incorporate the multi-prep specimen processing method, thereby making it possible to use a single processed specimen to screen for all three viruses.