Multicenter Performance Evaluation of a New TaqMan PCR Assay for Monitoring Human Immunodeficiency Virus RNA Load

A TaqMan real-time PCR assay, the COBAS TaqMan human immunodeficiencyvirus (HIV) (HPS-CTMHIV) PCR assay, recently developed for thequantification of HIV type 1 RNA in plasma, was evaluated incomparison with the licensed COBAS AMPLICOR HIV-1 MONITOR (CAHIM)assay. In this study, we have analyzed the tests' sensitivities,precisions, and linearities using multiple replicates of a standardpanel of HIV RNA covering a 7-logarithm range of concentrations,as well as serial threefold dilutions of high-titer clinicalsamples. The subtype inclusivity was also investigated, usinga panel of subtypes A to H, while a collection of 160 clinicalsamples was analyzed to assess the tests' specificities andthe systems' similarities. The results of these experimentsshowed that the HPS-CTMHIV assay has a sensitivity of 53 copies/ml(95% hit rate), 100% specificity, and good intra- and interassayprecision. The results of the HPS-CTMHIV assay were linear inthe 50- to 10⁷-copies/ml range, with a correlation coefficient(R) for expected versus observed results of 0.98. Compared tothe CAHIM assay, the HPS-CTMHIV assay showed a high correlation(R = 0.99) across the dynamic range of RNA concentrations that,for the CAHIM assay, requires two different sample preparations.Equivalent performances were also observed for the two systemsin the detection and quantification of HIV subtypes A to H.These data indicate that the HPS-CTMHIV assay may be one ofthe tests of choice for monitoring viral load throughout thecourse of HIV infection and during highly active antiretroviraltherapy.

INTRODUCTION

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Introduction

The quantification of human immunodeficiency virus type 1 (HIV-1)RNA in plasma has revolutionized the management of infectedpatients. This parameter is the main prognostic factor for evaluatingthe progression of the disease and for timing the initiationof treatment and monitoring its efficacy (1, 5, 7, 10, 11, 14,15, 16). Commercially available assays for the quantificationof HIV-1 viral RNA use a variety of techniques, from reversetranscriptase PCR to the branched-DNA assay (2, 12, 20, 21).

The COBAS AMPLICOR HIV-1 MONITOR (CAHIM) test v. 1.5, a quantitativereverse transcriptase PCR assay (3), has been widely used inresearch as well as in clinical practice. Quantification ofHIV-1 RNA with this assay requires two sample preparation procedures:in the first, ultrasensitive method, 500 µl of plasmais centrifuged to increase the input of plasma RNA to achievea quantitative range of 50 to 2,000 copies/ml of plasma, whereasin the second, standard protocol, with a starting plasma volumeof 200 microliters, only an amount of RNA corresponding to 25microliters of plasma is introduced in the PCR, resulting ina dynamic range of 500 to 500,000 copies/ml. A single-step techniquewith a wider dynamic range is therefore desirable for routinelaboratory applications. The real-time PCR technology alreadydeveloped for several noncommercial viral assays offers notonly broader quantification ranges but also faster turnaroundtime and more effective prevention of contamination (due toa closed-tube configuration) (4, 13, 23).

In this investigation, we compared the linearity, reproducibility,and sensitivity of the COBAS AMPLICOR HIV-1 MONITOR test v.1.5 with those of a newly developed real-time PCR assay, theCOBAS TaqMan HIV (HPS-CTMHIV) assay, which has a semiautomatedformat and dedicated hardware and software.

MATERIALS AND METHODS

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Materials and Methods

In this multicenter trial, we evaluated the performance characteristicsof the HPS-CTMHIV PCR assay, a semiautomated real-time PCR testbased on a dual-labeled hybridization probe targeting the gagregion. After manual HIV RNA extraction using the High Purekit (Roche Diagnostics, Mannheim, Germany), which consists ofsubsequent steps of lysis and glass particle RNA capture andpurification, the TaqMan assay is performed using a dedicatedCOBAS TaqMan 48 instrument and the results are expressed innumbers of HIV RNA copies/ml. The prevention of carryover contaminationand the integrity of the sample are ensured by the use of AmpEraseand by an internal control (quantitation standard) (8, 17).

All experiments were performed at sites 1, 2, and 3. The sensitivity,specificity, linearity, and subtype performances were analyzedin comparison with those of the CAHIM, a quantitative PCR assaylicensed for diagnostic use (3). The HPS-CTMHIV assay and theCAHIM assay were used in accordance with the manufacturer'sinstructions included in the test package inserts.

Precision, sensitivity, and specificity. Intra- and interassay reproducibilities were investigated usingan eight-member standard HIV RNA panel with titers ranging from30 to 5 x 10⁶ copies/ml (AcroMetrix, Benicia, CA). Six replicatesof each standard panel member were analyzed on three consecutivedays. These experiments were also used to estimate the assay'slimit of detection, calculated by probit analysis, while thespecificity was assessed by testing 100 samples obtained fromhealthy donors or patients with HIV-unrelated diseases.

Linearity and dynamic range. Linearity and dynamic range were investigated using 13 threefoldserial dilutions and an undiluted sample of a 5 x 10⁶-copies/mlconcentration of the AcroMetrix HIV DNA panel member as wellas of a high-titer clinical specimen selected at the trial sites.Each dilution was run in triplicate.

Subtype identification. The HPS-CTMHIV assay's ability to detect and quantify differentsubtypes with the same efficiency was analyzed using a standardpanel consisting of eight members, representing the group Mclade, subtypes A to H, tested in triplicate on three consecutivedays (HIV-1 RNA clade performance panel PRD201; Boston BiomedicaInc.).

Comparison of assays. Parallel experiments were performed at one of the trial sites(site 1) with the CAHIM assay, a quantitative PCR assay witha 4-log dynamic range of concentrations of 50 to 750,000 copies/mlobtained by combining two different sample preparation procedures,the standard and the ultrasensitive (21). In addition, to comparethe main performance characteristics of the two systems, wealso analyzed their relative performances in quantifying 60clinical samples taken from patients with HIV infection anddifferent viral loads.

Both the HPS-CTMHIV assay and the CAHIM assay have been approvedby the European Union for in vitro diagnostic applications (CEmark) and are being reviewed by the FDA.

RESULTS

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Results

In this study, we report the results of a three-site multicenterevaluation of the HPS-CTMHIV assay, a real-time PCR system specificallydeveloped to improve the ease of use, turnaround time, and overallefficacy of current quantitative PCR assays.

Using an HIV RNA standard panel, we investigated the HPS-CTMHIVassay's precision and detection limit across a range of concentrationsfrom 10 to 10⁷ copies/ml. In several experiments, run on threeconsecutive days with six replicates of each panel member, weobserved a mean total precision (intra- and interassay reproducibility)of 56% (31% to 81% coefficient of variation) and a 95% detectionlimit of 53 copies/ml as estimated by probit analysis (Fig.1 and Tables 1 and 2). The assay results were linear over a5-log range from 10 to 10⁷ copies/ml, and the correlation betweenthe expected and observed results was very high, with an R of0.98 (Fig. 2). Similar findings were made when data obtainedwith the HPS-CTMHIV assay were compared to those produced withthe CAHIM assay using both the standard and the ultrasensitiveprocedure. Samples with titers higher than 10⁵ copies/ml hadto be further diluted, as they were above the upper limit ofthe CAHIM assay's reportable range (Fig. 3a). An excellent correlationbetween the two tests was also noted when we analyzed 72 clinicalsamples taken from patients with chronic HIV infection and awide range of viremia levels (Fig. 3b).

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FIG. 1. Intra- and interassay variability of the HPS-CTMHIV assay. Six replicates of an eight-member standard panel were tested by the participating laboratories on three consecutive days. CV, coefficient of variation; LOQ, limit of quantification.

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TABLE 1. Precision of HPS-CTMHIV test^a

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TABLE 2. Limit of detection as assessed by probit analysis^a

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FIG. 2. Linear correlation of observed versus expected results of HPS-CTMHIV test as assessed with 13 threefold serial dilutions and an undiluted sample of a 10⁶-copies/ml concentration of a standard panel member. R2, correlation coefficient.

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FIG. 3. Linear range correlation between the HPS-CTMHIV and the CAHIM tests as assessed with 13 threefold serial dilutions and an undiluted sample of a 10⁶-copies/ml concentration of a standard panel member (a) and with 72 clinical samples (b). R2, correlation coefficient.

We also used a collection of 100 samples obtained from healthydonors or patients with HIV-unrelated diseases to assess theHPS-CTMHIV assay's specificity. No HIV RNA was detected by eitherthe HPS-CTMHIV or the CAHIM assay, with 100% specificity (datanot shown). All major M clade subtypes, including subtypes Ato H, were accurately detected and quantified when analyzedat two different concentrations, 300 and 3,000 copies/ml (Fig.4a and b).

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FIG. 4. Identification of subtypes A to H by the HPS-CTMHIV assay as assessed by analyzing an eight-member standard panel diluted to a nominal concentration of 3 x 10² copies/ml (a) and 3 x 10³ copies/ml (b). HIV RNA log concentrations were assessed using the HPS-CTMHIV and the CAHIM tests and compared to the titers calculated by the panel manufacturer (BBI CA, Boston Biomedica Inc., MA).

DISCUSSION

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Discussion

The HPS-CTMHIV test has been developed to standardize the real-timetechnique based on TaqMan instruments and to improve the performanceof the existing PCR assays (13). Based on this evaluation, theHPS-CTMHIV assay appears to have achieved these goals, confirmingits expected wide dynamic range and increased sensitivity. Ourdata indeed show that the assay can quantify concentrationsranging from 10 to 10⁷ copies/ml, thus including and extendingthe range obtainable by the CAHIM test, the FDA- and CE-approvedtest we used for system comparison. However, the CAHIM assaymakes use of two different sample preparation procedures, thestandard and the ultrasensitive. Specimens that are negativeby the former, at a detection limit of 400 copies/ml, have tobe reevaluated using the latter, which allows for a range of50 to 5 x 10⁴ copies/ml (21). Although laboratory managers maydecide beforehand which preparation to use based on the expectedHIV titers of the sample, the possibility of avoiding repeattesting by using the HPS-CTMHIV test instead of the CAHIM testrepresents a significant advantage in terms of cost effectivenessand workflow.

The HPS-CTMHIV assay's precision, as assessed by intra- andinterassay reproducibility, is good and is comparable to thatof the CAHIM assay. Further improvement is expected when theRNA extraction procedure, presently labor-intensive and lengthy,is automated (6). Medium-sized to large laboratories, with highsample turnover, may indeed benefit from fully automated samplepreparation using dedicated instrumentation (6, 18). In thisrespect, the COBAS AmpliPrep instrument, a nucleic acid extractoralready in use in conjunction with the CAHIM (COBAS AmpliPrep/COBASAMPLICOR HIV MONITOR) reagents, is being validated as the front-endinstrument for the HPS-CTMHIV test (19).

The issue of potential carryover has already been solved forthe CAHIM assay through pretreatment with uracyl-N-glycosylase,the enzyme that specifically inactivates dUTP-containing amplicons.This step is also included in the HPS-CTMHIV assay, with additionalsafety derived from the closed-tube procedure typical of thehydrolysis probe technology used with TaqMan instruments.

This investigation was not designed to evaluate the efficacyof HIV-1 RNA quantification for the various non-B subtypes,since only two dilutions of a single strain corresponding tothe most frequent HIV-1 subtypes were tested. However, in thisstudy we show that both tests are able to detect the subtypestested at low HIV RNA levels (300 copies/ml) and provide similarresults at the higher titers tested (3,000 copies/ml). Additionalstudies of larger numbers of clinical samples are warrantedto confirm these data and to assess the ability of the HPS-CTMHIVassay to detect newly emerging strains and recombinants (9,22).

In conclusion, we have shown that the HPS-CTMHIV test providesperformances similar to those of the licensed CAHIM test withregard to specificity, sensitivity, and precision and has additionaladvantages in terms of a larger dynamic range, high throughput,and absence of carryover during the PCR amplification procedure.

ACKNOWLEDGMENTS

COBAS, TaqMan, AmpliPrep, AMPLICOR, High Pure, and AmpEraseare trademarks of Roche.

FOOTNOTES

* Corresponding author. Mailing address: Roche Molecular Diagnostics, Roche Instrument Center AG, Lettenstrasse, 6343 Rotkreuz, Switzerland. Phone: 41 41 7992815. Fax: 41 41 7992845. E-mail: giuseppe.colucci.gc1{at}roche.com.

Published ahead of print on 27 September 2006.

REFERENCES

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