Maurice Rosenstraus

Enhancing the specificity of the COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae by retesting specimens with equivocal results.

ABSTRACT The COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeaecross-reacts with certain strains of nonpathogenicNeisseria species. In some strains, the target sequence is identical to that of N. gonorrhoeae, whereas other strains have a small number of mismatches within the regions recognized by the primers or probe used in the COBAS AMPLICOR NG test. These cross-reactive strains are occasionally present in urogenital specimens, causing false-positive results in the COBAS AMPLICOR NG test. Analysis of the data generated in a large multicenter clinical trial showed that 2.9% of the specimens gave signals betweenA660s of 0.2 and 3.5 but that one-half of these equivocal specimens did not contain N. gonorrhoeae. Most of these equivocal specimens were correctly classified as true positive or true negative by retesting in duplicate and defining a PCR-positive result as two of three results with an A660 of ≥2.0. If specimens had been classified as positive or negative based on a single test result using a cutoff of anA660 of 0.2, specificity would have ranged from 96.2 to 98.9% depending on specimen type, sex, and presence of symptoms. By employing the equivocal zone-retesting algorithm, specificity increased to 98.6 to 99.9% with little effect (0.1 to 4.9% decrease) on sensitivity in most specimen types, enabling the test to achieve a positive predictive value of at least 90% in populations with a prevalence of 4% or higher. In lower-prevalence populations, the test could be used to screen for presumptive infections that would have to be confirmed by an independent test.

Performance Characteristics of a Quantitative, Homogeneous TaqMan RT-PCR Test for HCV RNA

We developed a homogeneous format reverse transcription-polymerase chain reaction assay for quantitating hepatitis C virus (HCV) RNA based on the TaqMan principle, in which signal is generated by cleaving a target-specific probe during amplification. The test uses two probes, one specific for HCV and one specific for an internal control, containing fluorophores with different emission spectra. Titers are calculated in international units (IU)/ml by comparing the HCV signal generated by test samples to that generated by a set of external standards. Endpoint titration experiments demonstrated that samples containing 28 IU/ml give positive results 95% of the time. Based on these data, the limit of detection was set conservatively at 40 IU/ml. All HCV genotypes were amplified with equal efficiency and accurately quantitated: when equal quantities of RNA were tested, each genotype produced virtually identical fluorescent signals. The test exhibited a linear range extending from 64 to 4,180,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 21.6 to 30.4%, which implies that titers that differ by a factor of twofold (0.3 log10) are statistically significant (P = 0.005). The test did not react with other organisms likely to co-infect patients with hepatitis C and exhibited a specificity of 99% when evaluated on a set of samples from HCV seronegative blood donors. In interferon-treated patients, the patterns of viral load changes revealed by the TaqMan HCV quantitative test distinguished responders from nonresponders and responder-relapsers. These data indicate that the TaqMan quantitative HCV test provides an attractive alternative for measuring HCV viral load and should prove useful for prognosis and for monitoring the efficacy of antiviral treatments.

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

BACKGROUND: 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.

Simultaneous extraction of hepatitis C virus (HCV), hepatitis B virus, and HIV‐1 from plasma and detection of HCV RNA by a reverse transcriptase‐polymerase chain reaction assay designed for screening pooled units of donated blood

BACKGROUND: Testing for viral nucleic acids should reduce the residual risk of transmitting viral infections by transfusion of blood components. The AmpliScreen Hepatitis C (HCV) Test, Version 2.0, was designed for screening pools composed of samples from individual units of blood or plasma.

Quality control of the polymerase chain reaction.

The polymerase chain reaction (PCR) has revolutionized both the basic and the applied aspects of the biomedical field with more than 40,000 papers having been published employing this technique. PCR has become an indispensable tool for basic research applications, such as cloning (1), sequencing (2), mRNA analysis (3), DNA and RNA quantitation (4-6), mutagenesis (7), and gene detection (8). Because of its ability to amplify minute quantities of nucleic acid specifically, PCR has also been applied with great success in clinical diagnostics (9-11), genetic testing (12-15), forensics (16,17), and environmental testing (18,19).

Ultrasensitive Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma by the AMPLICOR and COBAS AMPLICOR HIV-1 MONITOR™ Tests

The measurement of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels has become an important tool for identifying individuals likely to benefit from antiretroviral therapy (1-7) as well as monitoring patients on therapy (8-14), and is now regarded as standard medical practice for managing the treatment of HIV-1-infected individuals (15-21). Three commercially available assays for measuring HIV-1 RNA levels are available. The firstgeneration AMPLICOR HIV-1 MONITOR™ Test, which uses reverse transcriptase-polymerase chain reaction (RT-PCR) technology, has a lower limit of quantitation of 400 copies/mL of plasma (22,23). The NucliSens HIV-1 QT Test (Organon Teknika, Boxtel, Netherlands), a second-generation assay based on the nucleic acid sequence-based amplification technique, has a lower limit of quantitation of 100 HIV-1 RNA copies/mL of plasma (24). The Quantiplex HIV-1 Version 2.0 Test (Chiron, Emeryville, CA), which uses the branched-chain DNA signal amplification technique, has a lower limit of quantitation of 500 HIV-1 RNA copies/mL of plasma (25,26).

The AMPLICOR® HCV Tests for the Detection and Quantitation of Serum or Plasma HCV RNA

Clinical diagnosis of HCV infection is generally accomplished by using immunoserological assays to detect the presence of anti-HCV antibodies. Such immunoserological assays have been approved for blood donor screening, thereby reducing the incidence of post-transfusion hepatitis in the United States. Although useful, immunoserological assays have several limitations. Recent evaluations have shown that interpretation of these immunological tests often is difficult, since 25-90% (depending on the risk group under evaluation) of samples repeatedly reactive in the screening assay are negative on supplemental evaluation with a recombinant immunoblot assay (RIBA) (1,2). Also, the presence of anti-HCV antibodies indicates prior exposure to HCV infection, but cannot be considered a marker for current infection. Nor can anti-HCV antibody levels be used to monitor response to therapeutic agents. Finally, in cases of acute HCV infection resulting from accidental needlestick exposure, many patients fail to produce antibody to HCV (3), which makes diagnosis of HCV infection impossible using immunoserological techniques. At the present time, an immunological assay for direct detection of HCV antigen is unavailable.