Comparative Evaluation of the Performance of the Abbott Real-Time Human Immunodeficiency Virus Type 1 (HIV-1) Assay for Measurement of HIV-1 Plasma Viral Load following Automated Specimen Preparation

The performance of the new Abbott real-time human immunodeficiencyvirus type 1 (HIV-1) assay for HIV-1 RNA load determinationin plasma was compared to that of the Abbott LCx HIV-1 RNA quantitativeassay following automated RNA isolation by the Abbott m1000extractor. The measured viral loads of 89 clinical specimensdiffered by mean 0.19 log₁₀ copies/ml (95% confidence interval,0.12 to 0.26 log₁₀ copies/ml). Although the difference in viralload determinations was positively skewed in favor of the LCxassay, it did not reach statistical significance (P = 0.42).Results were linearly associated (R² = 0.94) and strongly correlated(R = 0.96). Good performance was observed with HIV-1 subtypesother than B and circulating recombinant forms, although resultsobtained with two subtype G specimens and one H specimen showeda more substantial difference.

INTRODUCTION

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Introduction

Since its introduction in the 1990s, measurement of plasma viralload (PVL) has become a cornerstone in the clinical managementof human immunodeficiency virus type 1 (HIV-1) infection. Thecentral role played by PVL has considerably increased laboratoryworkload, making necessary an improvement in technology. A varietyof commercial assays are available for the measurement of HIV-1PVL. Among them, real-time PCR is the latest development, offeringmany advantages over traditional molecular methods (3, 7, 11).These include (i) decreased performance time attributed to reducedcycle time, reduced amplicon size, and the elimination of anadditional step needed for product detection; (ii) increasedsensitivity as a result of the employment of fluorescence detectionmethods; (iii) decreased carryover contamination due to theuse of a close system for the amplification and detection; and(iv) wider dynamic range. Further enhancements have been achievedby the introduction of automated nucleic acid extractions, resultingin completely automated assays with an average turnaround timeof 2 h.

The objective of this study was to evaluate the performanceof the new Abbott real-time HIV-1 assay (referred to as thereal-time assay) for PVL quantitation in comparison with theAbbott LCx HIV-1 RNA quantitative assay (referred to as theLCx assay). Both assays target the integrase region of the HIV-1genome. In the LCx assay, 24 samples can be processed in onerun, including 21 clinical specimens and 3 controls. The rangeof quantification is from 50 to 1 million copies/ml. In thereal-time assay, 48 samples can be processed in one run. Therange of quantification is 40 to 10 million copies/ml. Previousstudies have assessed the performance of the LCx assay and foundthe test suitable for the management of patients infected byHIV-1 group M subtypes (1, 4, 8, 9).

MATERIALS AND METHODS

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Materials and Methods

Patient and samples. Samples were randomly collected from 92 HIV-1-seropositive patientsattending the Ian Charleson Day Centre clinic at Royal FreeHospital in London. Plasma was prepared from EDTA-anticoagulatedblood and frozen at –80°C within 6 h of collection.

Viral load determination. Plasma RNA was extracted and concentrated using the Abbott m1000instrument, which uses magnetic particle technology to capturenucleic acid extracted from 1 ml of plasma. Each sample wasexamined in parallel using the LCx HIV-1 quantitative assayand the Abbott real-time HIV-1 assay according to the manufacturer'sspecification.

HIV-1 subtyping. In 51 patients, with PVLs of >1,000 copies/ml and for whichsufficient sample was available, HIV-1 subtype was determinedfrom pol gene sequences obtained by the Viroseq kit (CeleraDiagnostics). Briefly, total RNA was manually extracted fromplasma, and the protease region and two-thirds of the reversetranscriptase region were amplified by reverse transcription-PCRand sequenced before being analyzed on a 3100-Avant geneticanalyzer (Applied Biosystems, United Kingdom). Subtypes weredetermined by phylogenetic analysis employing Clustal X forsequence alignment and PAUP version 4 (Sinauer Associates) togenerate a phylogenetic tree. Reference sequences were derivedfrom the Los Alamos database (www.lanl.gov).

Statistical analysis. Plasma HIV-1 RNA levels were log₁₀ transformed before beingsubjected to statistical analysis. Averages were statisticallycompared by using the paired z test. Linear regression and correlationanalysis were employed to determine assay relationship. Themethod of Bland and Altman was applied in order to assess theagreement between the two assays.

RESULTS

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Results

A total of 92 plasma samples were tested in parallel by theLCx assay and the real-time assay, including 67/92 (73%) samplesfrom antiretroviral-drug-naïve patients and 25/92 (27%)samples from patients on highly active antiretroviral therapy(HAART). Overall, 63/92 (68%) samples were quantified by bothassays, with viral loads ranging from 1.7 log₁₀ to 6 log₁₀ copies/mlfor the LCx assay and 1.6 log₁₀ to 6.4 log₁₀ copies/ml for thereal-time assay.

There were 14/92 (15%) samples undetectable by both assays.These included 13/14 samples from patients on stable HAART and1/14 from a drug-naïve long-term nonprogressor. The lattersample also showed a viral load of <50 copies/ml when testedby the Cobas-Monitor 1.5 assay (Roche Diagnostics, Germany).Discrepant results at the lower limit of detection included10/92 (11%) samples showing viral loads between 63 and 251 copies/mlby the LCx assay but <40 copies/ml by the real-time assay.Conversely, 2/92 (2%) samples showed viral loads of 1,200 and158 copies/ml, respectively, by the real-time assay but <50copies/ml by the LCx assay.

Finally, 3/92 samples (3%) were quantified by the LCx assayat levels of 284 –11,300 copies/ml but were inhibitoryby the real-time assay. As there was insufficient sample forrepeating testing, these samples were excluded from furtheranalysis.

The two assays showed a high degree of correlation (Fig. 1).The linear regression equation for log₁₀ HIV RNA copies permilliliter was y = 0.96x – 0.007 (R² = 0.94). Viral loadsobtained by the two assays differed on average by 0.19 log₁₀(95% confidence interval, 0.12 to 0.26 log₁₀ copies/ml). Thedifference was not significant as determined by the paired ztest (P = 0.42). Agreement between the two methods was calculatedby the method of Bland and Altman by plotting differences againstthe average (Fig. 2). One of 92 (1%) samples was out of the95% interval of agreement, with a difference of –1.4 log₁₀copies/ml (<50 copies/ml by LCx and 1,259 copies/ml by thereal-time assay). The sample was from a patient on HAART infectedwith HIV-1 subtype B.

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FIG. 1. Correlation of viral load results obtained with 87 plasma samples tested in parallel by the Abbott LCx HIV-1 RNA quantitative assay and the Abbott real-time HIV-1 assay. The linear regression trend is shown. The lowest limit of detection was adjusted to 1.7 log₁₀ copies/ml for both assays.

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FIG. 2. Agreement of plasma viral load determinations obtained by the Abbott LCx HIV-1 RNA quantitative assay and the Abbott real-time HIV-1 assay. The dotted horizontal lines correspond to the 95% limit of agreement, given by the mean difference plus or minus twice the standard deviation of the difference. The sample outside the 95% interval was obtained from a patient infected with HIV-1 subtype B. SD, standard deviation.

Table 1 summarizes results obtained by the two assays from 51samples with known HIV-1 subtypes. The coefficient of correlation(R²) between the two assays was 0.94 for subtype B and 0.91for genotypes other than B. The difference in PVL quantitationwas not significant overall, although results obtained withtwo subtype G specimens and one H specimen showed a more substantialdifference (Table 1). The amplification curved did not showany grade of inhibition for these three samples in the real-timeassay. The samples were insufficient for further investigation.

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TABLE 1. Comparison of HIV-1 plasma viral loads obtained by the Abbott LCx HIV-1 RNA quantitative assay and the Abbott real-time HIV-1 assay, according to the infecting HIV-1 subtype

DISCUSSION

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Discussion

This study showed an excellent correlation and a high degreeof concordance between the new assay, Abbott real-time HIV-1assay and the Abbott LCx HIV RNA quantitative assay, with allexcept one differences falling within the 95% interval of agreement.The one divergent sample showed a PVL of 1,200 copies/ml bythe real-time but <50 copies/ml by the LCx assay. The samplehad been collected from a patient on HAART who was infectedwith HIV-1 subtype B. In both assays the sample met the qualitycontrol criteria to be considered and acceptable result, thenegative, low and high positive controls were within the acceptablerange and the internal controls were not inhibited.

The differences in PVL determination were positively skewed,indicating that the viral load estimations obtained by the LCxassay were overall higher than those obtained by the real-timeassay. However, the difference did not reach statistical significance.These observations indicate that the real-time assay can beused for those patients previously managed by the LCx assay.

There were 14 samples that showed undetectable PVL by both assays.Of these, 13 were from patients on stable HAART with undetectableplasma viremia documented for at least one year. One drug-naïveblack African patient with undetectable viral load was a long-termnonprogressor with a CD4 cell count consistently above 1,000cells/mm³, suggesting that he may have low levels of virus replication.The patient also showed an undetectable viral load by the RocheCobas-Monitor 1.5 assay, which targets a different region ofthe HIV-1 genome (gag gene).

Reliable performance at the lower limit of detection is a criticalfeature of PVL determination. The prevalence of discrepanciesat this level was 13%, in most cases represented by samplesthat gave low-level PVL measurements in the LCx assay (61 to251 copies/ml) but were <40 copies/ml in the real-time assay.Of note, all these were samples from patients on stable HAARTand with longstanding viral suppression (<50 copies/ml).We conducted a reproducibility analysis of PVLs greater than50 copies/ml but less than 400 copies/ml obtained by the LCxassay in patients with histories of suppressed viral loads whileon stable HAART. Results showed that only 8/32 (25%) resultsof the PVL were reproduced in repeated testing (data not shown).This finding suggests that there may be a greater degree oferror for the LCx assay at the lower limit of detection thanfor the real-time assay.

As HIV-1 infection continues to spread, its genetic diversificationincreases worldwide. Subtype-related variability in the performanceof viral load assays has been widely reported (2, 6, 8, 10).Previous studies have demonstrated that the high degree of nucleotideconservation within the targeted pol region enables the LCxassay to quantify HIV-1 subtypes A through G efficiently (4,8, 9). In this study, subtype-related variability in assay performancewas evaluated by using 51 specimens from patients infected withsubtype B, non-B subtypes A through H, and various circulatingrecombinant forms. Most specimens were quantified by the twoassays with comparable PVL measurements. A more substantialdifference was observed with samples from two patients infectedwith subtype G and one patient infected with subtype H whichshowed lower PVL levels by the real-time assay than by the LCxassay. Given the limited number of samples, the significanceof this observation is unclear. In London, 42% of newly diagnosedHIV-1-infected patients carry non-B subtypes or recombinantforms, including 2.5% with subtype G and approximately 0.5%with subtype H. Although the prevalence of subtypes G and His generally low in Europe, with both subtypes being found mainlyin Central Africa, subtype G is among the most prevalent non-Bsubtypes found in some European countries, such as Spain andPortugal (5). Using a reference HIV-1 subtype panel, includingsubtypes G and H, Braun et al. reported excellent performanceof the real-time assay with PVLs higher than those measuredby the Bayer bDNA and Roche Amplicor Monitor v1.5 assays (P.Braun, R. Ehret, F. Zabbai, S. Thamm, S. Schaffer, and H. Knechten,Eur. Meet. Mol. Diagn.). Thus, while this finding indicatesthat the real-time assay can be considered appropriate for themanagements of patients infected in many parts of the world,ongoing vigilance is recommended to evaluate assay performancewith existing and emerging divergent strains.

FOOTNOTES

* Corresponding author. Mailing address: Department of Virology, Royal Free Hospital and Royal Free and University College Medical School, Royal Free Hospital, Pond Street, London NW3 2PF, United Kingdom. Phone: 44-20 77940500. Fax: 44-20 7830 2854. E-mail: a.geretti{at}medsch.ucl.ac.uk.

REFERENCES

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