Sherrol Hoffa McDonough

Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

ABSTRACT Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications.

Significant Closure of the Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Preseroconversion Detection Windows with a Transcription-Mediated-Amplification-Driven Assay

ABSTRACT While the present generation of serology-based assays has significantly decreased the number of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections acquired by transfusion, the possibility of infected donations escaping detection still exists. The average seronegative viremic window duration during which immunological assays are unable to detect the virus is estimated to be between 16 and 22 days for HIV-1 and approximately 70 days for HCV. Significant reduction of detection window duration was demonstrated using a nucleic acid amplification assay, the Procleix HIV-1/HCV Assay, which utilizes transcription-mediated amplification technology to simultaneously detect HIV-1 and HCV RNAs. For 26 commercially available HIV-1 seroconversion panels tested, specimens were reactive in the HIV-1/HCV assay at the same time as or earlier than in serological assays. Overall, the HIV-1/HCV assay was able to reduce the detection window duration by an average of 14 days and 6 days compared to tests relying on recognition of HIV-1 antibody and p24 antigen, respectively. For 24 commercially available HCV seroconversion panels tested, the specimens were reactive in the HIV-1/HCV assay at an earlier blood sampling date than in serological assays, reducing the detection window duration by an average of 26 days. Similar results were obtained in testing the HIV-1 and HCV seroconversion panels in the virus-specific HIV-1- and HCV-discriminatory assays, respectively. In conclusion, the HIV-1/HCV assay and corresponding discriminatory assays significantly reduced detection window durations compared to immunoassays.

Clinical specificity and sensitivity of a blood screening assay for detection of HIV-1 and HCV RNA

BACKGROUND: An HIV‐1 and HCV NAT blood screening assay (Procleix HIV‐1/HCV, Gen‐Probe, Inc.) simultaneously detecting HIV‐1 and HCV RNA) has been implemented. Donor plasma samples reactive in the Procleix HIV‐1/HCV assay are tested with the HIV‐1 and HCV discriminatory assays to resolve whether HIV‐1 RNA, HCV RNA, or both are present.

High Throughput Assay for the Simultaneous or Separate Detection of Human Immunodeficiency Virus (HIV) and Hepatitis Type C Virus (HCV)

© 1998 S. Karger GmbH, Freiburg Fax (07 61) 4 52 07 14 www.karger.com Received: December 2, 1997 Accepted: March 9, 1998 S. H. McDonough Gen-Probe Incorporated 10210 Genetic Center Drive San Diego, CA 92121 (USA) Fax +1 619 41 088 71 This article is also accessible online at: http://BioMedNet.com/karger High Throughput Assay for the Simultaneous or Separate Detection of Human Immunodeficiency Virus (HIV) and Hepatitis Type C Virus (HCV) S. H. McDonough C. Giachetti Y. Yang D. P. Kolk E. Billyard L. Mimms

Sensitivity of the Procleix HIV-1/HCV Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA in a High-Risk Population

ABSTRACT The Procleix HIV-1/HCV Assay is a high-throughput nucleic acid test for the simultaneous detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNA during blood donor screening. This study evaluated the clinical sensitivity of the Procleix assay and assessed the assays ability to identify HIV-1- and HCV-infected individuals undetected by standard serologic tests. Plasma samples were obtained prospectively from 539 individuals at high risk for HIV-1 and HCV infection at seven clinics affiliated with Johns Hopkins University. Samples were tested in the Procleix HIV-1/HCV Assay and, if reactive, were then tested in the Procleix HIV-1 and HCV discriminatory assays to differentiate the source of viral RNA positivity. Of these 539 subjects, 287 (53.2%) tested reactive in the Procleix HIV-1/HCV Assay. In discriminatory assay testing, 12 of 287 subjects (4.2%) were reactive for HIV-1 RNA only, 260 (90.6%) were reactive for HCV RNA only, and 11 (3.8%) were coinfected with HIV-1 and HCV. The clinical sensitivity for samples tested neat was 100% for HIV-1 and 99.3% for HCV. Three subjects with Procleix HCV reactive/seronegative results seroconverted upon follow-up and were confirmed as Procleix HCV yield cases. The Procleix HIV-1/HCV Assay is a highly sensitive test that detects ongoing and early HIV-1 and HCV infection in a significant number of subjects at high risk for these diseases. Confirmation of Procleix yield cases upon follow-up demonstrated the ability of the Procleix HIV-1/HCV Assay to detect the presence of HIV-1 and HCV in blood earlier than standard serologic tests.

Application of Transcription-Mediated Amplification to Quantification of Gene Sequences

To be useful in clinical settings, hybridization-based assays must have high specificity and adequate sensitivity to detect clinically relevant concentrations of nucleic acid, and must be easy to use. Processing of patient specimens for these assays should involve a minimal number of steps so that the entire assay procedure will fit easily into the clinical laboratory.