Amplicor assay

Fig. 2.7 Virologic response of BILN 2061 (200 mg, twice a day) in pretreated and drug naive patients with minimal fibrosis. Upper and lower yellow lines represent the limits of detection of the Amplicor assay. Upper black triangles represent administration of BILN 2061.

It is not the aim of this chapter to describe the procedure for HIV-1 viral load determination using the Amplicor assay. The procedure for performing this assay is described well in the kit insert provided by the manufacturer. 

A key consideration with molecular assays is controlling for inhibition of amplification. This is particularly critical for the detection of MTb nucleic acid from respiratory specimens, because these specimen types often contain blood or glycoprotein, which can inhibit amplification. The Amplicor assay contains an internal control, and a negative result cannot be reported unless the internal control is detected. The MTD test does not contain an inhibition control, though many laboratories include such a control by adding a positive control material to a second aliquot of the clinical specimen. 

Nkengasong JN, Bile C, Kalou M, Maurice C, Boating E, Sasson-Moroko M> et al. Quantification of RNA in HIV Type 1 subtypes D and G by NucliSens and Amplicor Assays in Abidjan, Ivory Coast. AIDS Res Hum Retroviruses 1999 15 495-8. 

Revets, H., etal. (1996). Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICOR-HTV Monitor, and QUAN l lPLEX HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol 34,1058-1064. 

Three commercial assays for viral load determination are currently available Quantiplex (Chiron), NASBA (Organon-Teknika), and Amplicor (Roche). Only the latter would fall into the topic of this chapter (see Note 1), because it is based on the RNA- or reverse transcriptase-polymerase chain reaction (RT-PCR) technique. 

Molecular assays have utility for the diagnosis of active GMV disease, because GMV DNA concentrations are higher in patients with active GMV disease than in those with asymptomatic infection. A study in fiver transplant recipients using the Amplicor PGR assay showed that the median peak viral load in patients with asymptomatic infection was 1850 copies/mL compared with 55,000 copies/mL for those with active disease. The viral load cutoff that was most predictive of the development of active disease was between 2000 and 5000 copies/mL of plasma. A similar study in renal transplant recipients using the Hybrid Gapture assay showed that the risk of developing GMV disease increased from 1.5% with a viral load of 10,000 copies/mLof bloodto 73% when the viral load was 1 million copies/mL of blood. It is important to note that the viral load cutoffs differed in the two studies because of the use of different assays and differences in specimen type (plasma versus whole blood). 

Both the MTD test and the Amplicor test have a sensitivity of 95% to 98% when using APB smear-positive respiratory specimens, and the specificity ranges from 99% to 100%. However, early studies showed that the sensitivity for APB smear-negative specimens was -50%. Based on these data, it was clear that the test could not he used to rule out MTb infection on smear-negative respiratory specimens. This further limited the clinical utility of these tests, because it became clear that the nucleic acid testing would be used to supplement the AFB smear and culture rather than replace these testing modalities. Another limitation of the currently available nucleic acid assays is that they can be used only on specimens from patients who had not received antituberculosis therapy within the past 12 months. This limitation was included because DNA can persist in respiratory secretions (and other body fluids) for months after the mycobacteria are no longer viable. 

Several qualitative assays have been approved by the FDA for the detection of HCV RNA in plasma or serum specimens (Table 42-7). The AmpHcor HCV test and the COBAS Amplicor HCV test (Roche Diagnostics, Indianapolis) are based on RT-PCR technology the COBAS test allows for... 

The intra-assay imprecision (SDs) of the HCV Versant bDNA and Amplicor RT-PCR assays ranges from 0.05 to 0.31ogio the Versant bDNA assay is more precise. The biological variation of HCV viral load ranges from 0.5 logio... 

Cahendo AM, St George K, Kao SY, Allega J, Tan BH> LaPontaine R, et al. Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients. J Clin Microbiol 2000 38 2122-7,... 

PCR-based assays that have been developed by Roche Molecular Systems (Pleasonton, CA, USA) are used for quantitative detection of HBV-DNA. Amplicor HBV monitor and its semiautomated version Cobas Amplicor HBV Monitor has lower detection limit of 1,000 copies/ml and 200 copies/ml respectively. 

Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use. 

---