Larry Mimms

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.

Discrimination of hepatitis B virus (HBV) subtypes using monoclonal antibodies to the PreS1 and PreS2 domains of the viral envelope.

We report the production and characterization of murine anti-PreS2 and anti-PreS1 monoclonal antibodies (mAb) and demonstrate their utility in discriminating hepatitis B virus (HBV) subtypes. On the basis of Western blotting and reciprocal competition binding to HBV virions, at least five distinct epitopes have been identified in the PreS domain: two within the PreS1 region and three within the PreS2 region. All PreS2 mAb bind M protein (gp33 and gp36) but only one group binds strongly to M and L proteins (p39 and gp42). This group determinant was mapped to peptide residues 120-145. The second group bound to an endoglycosidase F-sensitive epitope which is defined by a mannose-rich glycan at ASN 123 in the PreS2 region. The third group was mapped to peptide residues 150-174 and was reactive with the M envelope proteins but not L or S proteins on Western blots. All PreS1 mAb bind L protein but not M protein on Western blots. Using these mAb, HBV subtype assays were developed allowing evaluation of the Paris (1975) HBsAg subtype panel members along with other HBsAg-positive specimens. All Paris subtype members (except ayw2 and ayw3) could be easily distinguished by differential PreS2 mAb reactivity. The Paris subtypes, adw2, adw4, and adr, could be classified as distinct groups by PreS2 and PreS1 mAb binding. Specimens from Hong Kong and the United States classified as adw2 in the S region fell into two groups based on PreS2 mAb binding: one having reactivity similar to Paris adw2 subtype and the other having identical reactivity to Paris ayw1 subtype. Furthermore, some specimens classified as adr in the S region gave similar reactivity to the Paris ayr subtype in the PreS2 and PreS1 regions. One complicating factor in this approach toward subtyping was the discovery that some HBsAg positive sera may contain factors which block PreS epitopes. Grouping of HBV subtypes by PreS1, PreS2, and S mAb reactivity may allow better correlation with groupings based on HBV DNA sequence homology.

Epitope mapping of the PreS1 domain of the hepatitis B virus large surface protein

The large (L) surface glycoprotein of hepatitis B virus is an important component of the virion envelope derived from translation initiation at the 5 end of the PreS1 domain of the surface antigen open reading frame. Since key roles in virion assembly and infectivity have been postulated for this protein, further understanding of its structure and topology is important. To this end we have mapped the epitopes recognized by a panel of monoclonal antibodies specific for this polypeptide by examining their reactivity with a series of deletion mutants of the PreS1 region expressed in cultured cells. On the basis of this and other techniques, the antibodies fall into two groups mapping to two distinct epitopes spanning residues 27-35 and 72-78, respectively. Immunoprecipitation studies indicate that both regions are exposed on the surface of HBV-encoded particles.

HCV viral load in anti-HCV-reactive donors and infectivity for their recipients.

BACKGROUND:u2002 An attempt has been made to determine the minimum level of HCV nucleic acid in donors associated with infection of recipients. This is important for considerations about assay sensitivity, use of minipool versus single‐donation testing, and continued use of serologic testing.

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

Sensitive detection of genetic variants of HIV-1 and HCV with an HIV-1/HCV assay based on transcription-mediated amplification

This paper describes a comprehensive study of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genotype sensitivity of the transcription-mediated amplification (TMA)-based HIV-1/HCV assay, developed and manufactured by Gen-Probe Incorporated (San Diego, CA) for screening human plasma specimens in blood bank settings. The TMA HIV-1/HCV assay is a qualitative, in vitro nucleic acid testing system used for initial screening. HIV-1 and HCV discriminatory assays are used to distinguish between HIV-1 and HCV infection or co-infection. In this study, multiple unique specimens representing HCV genotypes 1-6 were tested at various dilutions. The results show that the TMA HIV-1/HCV assay and the TMA HCV discriminatory assay have similar HCV genotype sensitivity, as both assays detected all six genotypes at 100 copies/ml and nearly all replicates tested at 30 copies/ml. Similarly, numerous unique specimens representing HIV-1 group M subtypes (A-G), HIV-1 group N, and group O specimens were tested at various dilutions. The TMA HIV-1/HCV assay and the TMA HIV-1 discriminatory assay were found to have similar HIV-1 subtype sensitivity; all variants at 100 copies/ml and nearly all at 30 copies/ml were detected. These results indicate that the TMA assays meet the sensitivity requirements for blood screening in blood banks worldwide.

Quantitation of hepatitis B surface antibody by an automated microparticle enzyme immunoassay

A fully automated microparticle enzyme immunoassay, IMx AUSAB, was developed for the detection and quantitation of antibody against hepatitis B surface antigen (anti-HBs). The IMx AUSAB assay can complete 24 tests in less than 45 minutes. Anti-HBs concentrations in specimens are calculated automatically by comparison of the specimen rate to values determined from a stored standard curve. IMx AUSAB sensitivity is 2-3 mIU/ml, equivalent in sensitivity to AUSAB RIA or EIA. Specimens from blood donors, diagnostic and hospital patients, hepatitis B vaccinees, and individuals with a variety of infectious and autoimmune diseases tested in parallel by IMx AUSAB and AUSAB RIA or IMx AUSAB and EIA gave overall qualitative agreement of 97.8% (1265/1293) and 99.1% (1281/1293), respectively. The prevalence of anti-HBs ranged from 5.9% in volunteer blood donors to 47.0% of specimens from a sexually transmitted disease clinic. Most discordant specimens (18/34) were low level reactive (less than 10 mIU/ml) by AUSAB RIA, but negative by IMx AUSAB and AUSAB EIA. These specimens were also negative for antibodies to hepatitis B core antigen (anti-HBc). Six discordants were low level reactive by IMx but negative by RIA and EIA. Three of these six specimens were also reactive for anti-HBc suggesting that the IMx AUSAB reactivity resulted from the presence of low level anti-HBs. Quantitative agreement between IMx AUSAB and RIA or IMx and EIA for 106 specimens ranging in anti-HBs concentration from 1 to 30,000 mIU/ml gave linear correlation coefficients of 0.91 and 0.96, respectively. The IMx test was useful for monitoring hepatitis B vaccine response and seroconversion levels after hepatitis B infection.

Use of third-generation hepatitis C virus (HCV) enzyme immunoassay (EIA) to resolve second-generation HCV EIA-reactive and second-generation recombinant immunoblot assay-indeterminate blood samples: data to support current Food and Drug Administration guidance on HCV lookback

10 TRANSFUSION Volume 40, January 2000 On March 20, 1998, the Food and Drug Administration (FDA) issued a draft guidance document1 on hepatitis C virus (HCV) lookback. In this document, the FDA recommended that, before lookback-related consignee notification proceeds, further testing should be performed for cases involving donors who were identified as reactive by multiantigen second-generation HCV enzyme immunoassay (HCV EIA 2.0) and who were either not tested by a supplemental HCV assay or who tested indeterminate by the currently licensed second-generation HCV recombinant immunoblot assay (RIBA 2.0). Two options for further testing of these cases were presented. Option 1 applied when a stored donation sample was available which could be tested using the investigational third-generation HCV RIBA (RIBA 3.0) under an investigational new drug (IND) exemption. If the RIBA 3.0 was positive, consignee notification must proceed; if it was negative or indeterminate, consignee notification was not required or recommended. Option 2 involved the recall of the HCV EIA-2.0-reactive, RIBA 2.0-indeterminate donor and testing of a fresh blood sample from the donor by use of a currently licensed multiantigen screening test (e.g., third-generation HCV EIA [HCV EIA-3.0]). If the screening test was nonreactive, no further action was needed; if it was repeatedly reactive (RR), either a licensed or investigational supplemental HCV assay should be performed to resolve the donor’s status and determine whether lookback was warranted. Because retention samples exist for the majority of relevant HCV EIA 2.0-RR/RIBA 2.0-indeterminate donations (Stramer SS, oral communication, June 8,1998 and because donor recall is logistically difficult and often incomplete, further testing of the stored donation samples (Option 1 above) was clearly the preferred course of action for most blood centers. Unfortunately, investigational RIBA 3.0 kits were not available to support the testing stipulated above in the test volume required (e.g., the American Red Cross alone had over 10,000 HCV EIA 2.0-RR/RIBA 2.0-indeterminate donations that would require RIBA 3.0 testing). The lack of availability of RIBA 3.0 presented a serious problem to blood centers attempting to complete lookback activity in as timely a fashion as possible to avoid regulatory and/ or litigation problems. In response to this dilemma, several blood centers were considering triggering lookback on all HCV EIA 2.0-RR/RIBA 2.0-indeterminate donations, despite the knowledge that only 15 to 20 percent of these donations (or the donors) are predicted to test positive by RIBA 3.0, that less than 5 percent will test positive for HCV RNA, and prior donations by these donors have not been documented to be associated with recipient infection in lookback studies from Europe.2,3 Several sources of data suggested to us that the currently licensed HCV EIA 3.0 could serve as a test to triage these cases into those that do warrant potential triggering of lookback (i.e., HCV EIA 3.0-RR) and those that don’t (i.e., Use of third-generation hepatitis C virus (HCV) enzyme immunoassay (EIA) to resolve second-generation HCV EIA-reactive and second-generation recombinant immunoblot assay-indeterminate blood samples: data to support current Food and Drug Administration guidance on HCV lookback