Single-Point Mutations Causing More than 100-Fold Underestimation of Human Immunodeficiency Virus Type 1 (HIV-1) Load with the Cobas TaqMan HIV-1 Real-Time PCR Assay

Korn, Klaus; Weißbrich, Benedikt; Henke-Gendo, Cornelia; Heim, Albert; Jauer, Christin Mariel; Taylor, Ninon; Eberle, Josef

doi:10.1128/jcm.02204-08

Cited by 53 publications

(38 citation statements)

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“…There have been several reports of underquantitation of the viral load by CE-marked quantitative (diagnostic) HIV-1 NAT, traceable also to the assays' design [13,14,15,16]. These false-negative or underquantified test results occurred with screening or diagnostic NAT assays targeting 1 region of the HIV-1 genome in combination with new HIV-1 variants.…”

Section: Introductionmentioning

confidence: 99%

Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany

Chudy

Kreß

Halbauer

et al. 2013

Transfus Med Hemother

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Background: Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat). Methods: Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems. Results: Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening. Conclusion: HIV differs from other blood-borne viruses with regard to its fast evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1 variants.

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Section: Introductionmentioning

confidence: 99%

Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany

Chudy

Kreß

Halbauer

et al. 2013

Transfus Med Hemother

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“…However, the challenge posed by the continued diversification of the highly variable HIV genome in clinical isolates worldwide and its impact on testing is critically important and has increasingly become the focus of study. While most reports have emphasized underquantification of non-B subtypes (2,3,5,6,7,9), the discrepant samples in this study and others (2,10) showed that underquantification can also occur in subtype B and is not restricted to specific subtypes.…”

Section: Discussionmentioning

confidence: 47%

“…For samples containing similar sequence mismatches which have been previously analyzed by RGSP, the details of sample handling such as freeze/thaw cycles, age, and storage conditions of the samples are not available. These problems are compounded by the imperfect science of predicting effects of polymorphisms on PCR performance and the proprietary nature of the assay sequences, which require investigators to rely on manufacturer analysis (such as in this study) or to make assumptions about the primer and probe locations to interpret sequencing results (10). These factors pose a significant obstacle to achieving a rigorous understanding of effects of sequence variation on commercial HIV assays.…”

Section: Discussionmentioning

confidence: 98%

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Evaluation of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test and Identification of Rare Polymorphisms Potentially Affecting Assay Performance

2010

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We evaluated the FDA-approved Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 viral load assay for sensitivity, reproducibility, linearity, HIV-1 subtype detection, and correlation to the Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor). The limit of detection calculated by probit analysis was 23.8 copies/ml using the 2nd International WHO Standard and 30.8 copies/ml using Viral Quality Assurance (VQA) standard material. Serial dilutions of six patient samples were used to determine inter-and intra-assay reproducibility and linearity, which were very good (<8% coefficient of variation [CV]; between ϳ1.7 and 7.0 log 10 copies/ml). Subtype detection was evaluated in the CAP/CTM, Amplicor, and Bayer Versant HIV-1 bDNA 3.0 (Versant) assays using a commercially available panel. Versant averaged 0.829 log 10 copies/ml lower than CAP/CTM and Amplicor averaged 0.427 log 10 copies/ml lower than CAP/CTM for the subtype panel. Correlation with samples previously tested by Amplicor was excellent (R 2 ‫؍‬ 0.884; average difference [Amplicor value minus CAP/CTM value], 0.008 log 10 copies/ml). Of the 305 HIV samples tested, 7 samples generated CAP/CTM titers between 1.0 and 2.75 log 10 copies/ml lower than those for Amplicor. Three of these samples revealed primer and probe mismatches that could account for the discrepancies. Otherwise, the CAP/CTM assay exhibits excellent sensitivity, dynamic range, reproducibility, and correlation with Amplicor in an automated format.Measurement of HIV-1 RNA in the plasma of infected patients is critical for guiding treatment. Over the past decade, several commercial quantitative HIV-1 RNA assays have become available that utilize endpoint PCR, isothermal amplification, or signal amplification techniques. Most recently, Abbott Molecular (Des Plaines, IL) and Roche Molecular Systems (Branchburg, NJ) received FDA approval for their real-time PCR-based systems, the RealTime HIV-1 assay and Cobas AmpliPrep/Cobas TaqMan HIV-1 Test (CAP/CTM), respectively. Each assay has its own advantages and disadvantages in terms of sensitivity, equipment requirements, throughput, dynamic range, subtype detection, and cost (1a, 4, 6, 7, 8, 11, 13, 15, 16).The CAP/CTM test includes a "docked" version that permits automated "sample in-results out" analysis of specimens without user intervention. We evaluated this configuration and compared its performance to those of the Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor), and the Bayer Versant HIV-1 bDNA 3.0 assay (Versant). Seven samples which gave discrepant Amplicor versus CAP/CTM results were further evaluated by sequencing analysis. MATERIALS AND METHODS Limit of detection.To determine the limit of detection in the CAP/CTM assay, nine dilutions of the WHO reference material (HIV-1 RNA, 2nd International Standard, 97/650; NIBSC, Potters Bar, United Kingdom) or Viral Quality Assurance (VQA) standard (Rush University Medical Center, Chicago, IL) were prepared in Basematrix diluent (SeraCare Life Sciences, Milford, MA). Fourteen replicates a...

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“…The resulting evolution can be a challenge for design of molecular diagnostic assays that rely on reactivity of synthetic primers and probes with the target sequence. There are numerous reports in the literature that describe cases of underestimation of viral load in patients infected with virus that have divergent sequence in critical genome regions (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”

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confidence: 99%

Sequence Conservation of the Region Targeted by the Abbott RealTime HBV Viral Load Assay in Clinical Specimens

et al. 2013

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Single-Point Mutations Causing More than 100-Fold Underestimation of Human Immunodeficiency Virus Type 1 (HIV-1) Load with the Cobas TaqMan HIV-1 Real-Time PCR Assay

Cited by 53 publications

References 3 publications

Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany

Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany

Evaluation of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test and Identification of Rare Polymorphisms Potentially Affecting Assay Performance

Sequence Conservation of the Region Targeted by the Abbott RealTime HBV Viral Load Assay in Clinical Specimens

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