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Journal of Clinical Microbiology, April 1999, p. 1210-1212, Vol. 37, No. 4
Departments of Medicine and Clinical
Laboratories, North Shore University Hospital-NYU School of
Medicine, Manhasset, New York
Received 31 August 1998/Returned for modification 6 November
1998/Accepted 15 January 1999
This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was
detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and
in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although
the Quantiplex and NucliSens values were significantly different
(P < 0.001), with the NucliSens values being
approximately 0.323 log higher. Spearman correlation analysis indicated
that the overall changes in patient viral load patterns were highly
correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were
determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.
The measurement of human
immunodeficiency virus type 1 (HIV-1) RNA levels in plasma (viral load)
is presently one of the most valuable clinical tools for predicting HIV
disease progression (3, 4, 6, 9, 12, 14, 17, 20), for
determining the need to initiate or change antiretroviral therapy
(2, 10, 11, 17, 20, 21), and for evaluating the efficacy of
newly developed antiretroviral drugs (21). Several
commercial assays, employing different molecular technologies, for
measuring plasma HIV-1 RNA levels are available. These methods include
reverse transcriptase PCR, which is used in the Amplicor HIV-1
Monitor assay (Roche Diagnostic Systems, Branchburg, N.J.)
(16), branched-DNA (bDNA) techniques, which are
used in the Quantiplex HIV RNA assay (Chiron Diagnostics,
Emeryville, Calif.) (18, 23), and nucleic acid
sequence-based amplification, which is used in the NucliSens HIV-1 RNA
QT assay (Organon Teknika, Durham, N.C.) (13, 25, 26).
In addition to differences in the molecular bases of the
assays, certain critical test parameters, including the comparability of the actual HIV-1 RNA copy numbers detected and the lower limits of
assay sensitivity, can also vary significantly (13, 16, 18,
19, 23, 25, 26). The comparison of HIV-1 RNA values obtained from identical specimens by the various methods is important for two reasons. First, clinicians must often try to correlate viral
load results from new patients to previous viral load results obtained
by a different or less sensitive methodology. Second, clinical trial
studies of new antiretroviral agents use different assays to measure
responses to therapy. Equivalence of the RNA measurements would allow a
more accurate comparison of the overall responses to new therapy
regimens. Differences in HIV-1 RNA values could affect the total
log reduction in RNA levels, the percentage of patients who
achieve "undetectable" levels, and the determination of the
potential success or failure of new experimental treatment regimens. To
address this issue, we evaluated the NucliSens assay performance
characteristics, including assay sensitivity, reproducibility, and accuracy, and examined the relationship between NucliSens and Quantiplex viral load values.
The NucliSens assay performance characteristics were determined by
using HIV-1 RNA standards obtained from the AIDS Clinical Trials Group
Virology Laboratories Quality Assurance Program (VQA). The VQA
standards were serially diluted to yield the following standards
(numbers in parentheses denote the number of times each standard was
assayed over several independent test runs): 80 copies/ml (19), 100 copies/ml (20), 500 copies/ml (10), 1,000 copies/ml (8), 1,500 copies/ml (8), 3,000 copies/ml (20), 15,000 copies/ml (8), and 150,000 copies/ml (8). All standards
were quantitated with the NucliSens assay according to the
manufacturer's instructions.
Our laboratory was able to detect HIV-1 RNA in the 80-copy/ml standard
in 11 of 19 specimens tested (57.89%) and in the 100-copy/ml standard
in 17 of 20 specimens (85%). RNA was detected 100% of the time at all
other input levels tested. Assay reproducibility was evaluated by
determining the standard deviation from the mean log value obtained for
each of the standards after the multiple test runs. The standard
deviations for the standards tested are as follows: 80 copies/ml,
±0.322 log; 100 copies/ml, ±0.359 log; 500 copies/ml, ±0.182
log; 1,000 copies/ml, ±0.113 log; 1,500 copies/ml, ±0.164
log; 3,000 copies/ml, ±0.096 log; 15,000 copies/ml, ±0.101
log; and 150,000 copies/ml, ±0.067 log. The overall assay variation was ±0.1755 log, which was within the expected variation of
the assay in accordance with the published literature (25). Assay accuracy was evaluated by calculating the difference between the
input log value and the mean of the actual log values obtained for each
standard. The mean log differences from the expected log values are as
follows: 80 copies/ml, +0.117 log; 100 copies/ml, The relationship between NucliSens and Quantiplex HIV-1 RNA values
was determined by comparing the viral loads measured by both assays in
184 serial plasma samples collected, with Institutional Review Board
approval, from 14 patients participating in the protease inhibitor
nelfinavir (Agouron Pharmaceuticals, Inc., La Jolla, Calif.) phase III
clinical trials. In accordance with the nelfinavir clinical trial
protocol, bDNA levels were analyzed in duplicate 1-ml volumes with a
modified 1.0 version of the Quantiplex assay by Corning SciCor, Inc.
(Indianapolis, Ind.). NucliSens HIV-1 RNA measurements were
performed on site according to the manufacturer's instructions with an
initial 200-µl input volume (detection limit = 500 copies/ml). All samples undetectable at a 200-µl input volume were retested with a 1-ml input volume (detection limit = 100 copies/ml).
As shown in Table 1, the Quantiplex assay
detected HIV-1 RNA in 118 of 184 specimens, and the NucliSens assay
detected HIV-1 RNA in 171 of 184 specimens. With a 200-µl specimen
input volume, the NucliSens assay identified 22 of 37 samples
(59.46%) with HIV-1 RNA levels between 47 and 500 copies/ml. HIV-1 RNA
was detected in 15 additional specimens after the specimen input volume
was increased to 1 ml. All samples with RNA detectable by the
Quantiplex assay were positive by the NucliSens assay. The results
for 162 specimens (88.04%) were concordant based upon the detection
limits of 100 copies/ml for the NucliSens assay and 500 copies/ml for the Quantiplex assay (manufacturer's claim). There
were 22 samples with discordant results that had NucliSens values
of >500 copies/ml (mean value = 1,350 copies/ml), but
were reported as having <500 copies/ml by the Quantiplex assay.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparison of Levels of Human Immunodeficiency Virus Type 1 RNA
in Plasma as Measured by the NucliSens Nucleic Acid Sequence-Based
Amplification and Quantiplex Branched-DNA Assays
ABSTRACT
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0.118 log;
500 copies/ml, 0.026 log; 1,000 copies/ml, +0.008 log; 1,500 copies/ml, 0.120 log; 3,000 copies/ml, +0.006 log; 15,000 copies/ml, 0.063 log; and 150,000 copies/ml, 0.228 log. The
overall assay accuracy for the standards tested was 0.053 log. The
recovery of the input copies ranged from 94.10% for 100 RNA
copies/ml to 109.30% for 80 RNA copies/ml, with an overall mean recovery of 99.15% of the input RNA copies.
TABLE 1.
Comparison of HIV-1 RNA detection rates in serial plasma
samples obtained with the Quantiplex and NucliSens assays
A comparison of the absolute and log-transformed values, obtained by
the Wilcoxon signed-rank test, found a significant difference (P < 0.001) between the HIV-1 RNA values of the two
tests. The mean ratio of Quantiplex absolute values to NucliSens
absolute values was 1:2.441. The NucliSens RNA values were, on
average, 0.323 log higher than the Quantiplex values. Regression
analysis (Fig. 1) using the
log-transformed values determined that there was a significant linear
relationship between the Quantiplex and NucliSens values
(r = 0.912, P < 0.0001). The intercept of 0.30 was not significantly different from 0 (95% confidence interval, 0.01 to 0.61). The slope of 0.87 was significantly different from 1 (95% confidence interval, 0.80 to 0.95). This means that although the
two values correlate significantly, the NucliSens values are
consistently higher than Quantiplex values. Spearman correlation
coefficient analysis indicated that the overall changes in patient
viral load patterns were highly correlative between the two assays
(P < 0.001). These results were in agreement with several studies that found that the values obtained by the
first-generation nucleic acid sequence-based amplification assay and
the Amplicor reverse transcriptase PCR Monitor assay were most
often comparable but significantly different from the values obtained
with the Quantiplex assay (1, 5, 7, 8, 15, 19, 22, 24).
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The NucliSens assay was significantly more sensitive than the Quantiplex assay in detecting HIV-1 RNA, due to two factors. First, the lower limit of detection of the NucliSens assay was determined to be approximately 100 copies/ml (with a 1-ml input) based upon validation studies using VQA standards. Second, although a direct evaluation of the Quantiplex assay sensitivity was not performed, the correlation data suggests that the detection limit is actually in the range of 1,200 to 1,400 copies/ml. This would explain the 22 discrepant samples, which had NucliSens values of >500 copies/ml but were undetectable by the Quantiplex assay. The differences observed in this study, related both to the values of the two assays and the manufacturer's detection limits and to our estimated detection limit for the Quantiplex assay, may be related to methods of testing and quantitation. The NucliSens assay is based on target amplification, while the Quantiplex assay is based on signal amplification. The NucliSens results are extrapolated from the values obtained from three internal calibrators of known RNA copies coamplified with each test sample. Quantiplex results are based upon a calibration curve generated from external standards.
The successful use of highly active antiretroviral therapy in the treatment of HIV disease has demonstrated the need for accurate, ultrasensitive viral load assays to monitor the course of antiretroviral therapy. The ability to lower the detection limit by increasing the specimen input volume has proved to be a significant benefit of the NucliSens assay. The enhanced sensitivity of the NucliSens assay permitted the detection of a sustained rebound in viral load in 11 of the 14 patients on 15 occasions sooner than the Quantiplex assay. The rapid detection of increased viral replication could allow the earlier identification of patients on a failing regimen or indicate the need to supplement the regimen with an additional antiretroviral agent.
These studies clearly indicate that it is best to choose one viral load assay for monitoring patients and that the results from different assays should not be used interchangeably. One must be aware of the differences in the accuracy and sensitivity of the assays; all "undetectable" results are not equal. In addition, these differences must be taken into account when data from clinical trials of new antiretroviral agents is interpreted.
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ACKNOWLEDGMENTS |
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This study was funded in part by the Jane and Dayton Brown and Dayton Brown, Jr., Virology Laboratory and a grant from Organon Teknika Corp., Boxtel, The Netherlands.
We thank Dean Winslow of Agouron Pharmaceuticals for kindly providing Quantiplex viral load results, Joseph Romano for editorial review, and Nina Kohn for statistical analysis. We sincerely thank the nurses and volunteer patients from the North Shore University Hospital Center for AIDS Research and Treatment who faithfully participated in these studies.
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FOOTNOTES |
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* Corresponding author. Mailing address: North Shore-LIJ Health System Laboratories, 10 Nevada Dr., Lake Success, NY 11042. Phone: (516) 719-1079. Fax: (516) 719-1254. E-mail: cginocch{at}nshs.edu.
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Journal of Clinical Microbiology, April 1999, p. 1210-1212, Vol. 37, No. 4
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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