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Previous Article | Next Article 
Journal of Clinical Microbiology, November 1999, p. 3612-3614, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparison of the Quantiplex Version 3.0 Assay and a Sensitized
Amplicor Monitor Assay for Measurement of Human Immunodeficiency Virus
Type 1 RNA Levels in Plasma Samples
Helene C.
Highbarger,1
W. Gregory
Alvord,2
Min Kang
Jiang,1
Akram S.
Shah,1
Julia A.
Metcalf,3
H. Clifford
Lane,3 and
Robin L.
Dewar1,*
Virus Isolation Laboratory, Clinical Services
Program, Science Applications International
Corporation
Frederick,1 and Data
Management Services, Inc.,2 National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
Maryland 21702, and Laboratory of Immunoregulation, National
Institute of Allergy and Infectious Diseases, Bethesda, Maryland
208923
Received 23 March 1999/Returned for modification 13 May
1999/Accepted 23 July 1999
 |
ABSTRACT |
This study evaluated correlation and agreement between version 3 of
the Quantiplex human immunodeficiency virus type 1 (HIV-1) RNA assay
(v3 branched DNA [bDNA]) and a sensitized Amplicor HIV-1 Monitor
assay (reverse transcription [RT]-PCR) for the measurement of HIV
RNA. Three hundred eighteen samples from 59 randomly selected, HIV-1-seropositive persons on various drug protocols from the National
Institute of Allergy and Infectious Diseases HIV outpatient clinic were
studied. The results indicate that v3 bDNA and RT-PCR are highly
correlated (r = 0.98) and are in good agreement (mean difference in log10 copies/ml ± 2 standard
deviations = 0.072 ± 0.371). The relationship between values
obtained by both assays is given by the following equation:
log10v3 bDNA = 
0.0915 + 1.0052 · log10RT-PCR. This represents a 1.026-fold difference
between log10RT-PCR values and log10v3 bDNA values.
| |
INTRODUCTION |
The use of highly active
antiretroviral therapy in patients with human immunodeficiency virus
type 1 (HIV-1) infection has resulted in an increase in the number of
CD4+ T lymphocytes as well as in a reduction in plasma
virus load (2, 4-6). In order to assess the magnitude of
viral load reduction and the occurrence of rebound virus due to drug
failure or noncompliance, it is necessary to use as sensitive a viral
quantitation assay as is available. There are two widely used
techniques to achieve these ends: the Amplicor HIV-1 Monitor assay
(reverse transcription [RT]-PCR) and the Quantiplex HIV-1 RNA assay
(branched DNA [bDNA]). The Amplicor HIV-1 Monitor assay is an in
vitro nucleic acid target amplification (PCR) test for the quantitation
of HIV-1 RNA in human plasma, while the Quantiplex HIV-1 RNA assay is a
signal amplification nucleic acid probe assay for HIV-1 quantitation. The most current versions of these assays have a quantitative detection
limit of 50 copies (3, 7, 8, 11). Because patients may be
tested with both of these assays at some point in their treatment, it
is useful to determine how the resulting values are related to each
other and, ideally, to develop some formula for their comparison. We
hypothesize that tests designed to measure the same thing, albeit in
different ways, will show a quantifiable relationship to each other;
not only will they correlate, but they will show agreement. Previous
reports comparing Amplicor with Quantiplex version 2.0 have indicated
that although the results show a high correlation, they show poor
agreement (9, 10). In this study, we compared two of the
commercially available assays for the measurement of HIV RNA, the
Quantiplex version 3 assay (3) and a sensitized version of
the HIV-1 Amplicor Monitor assay (7, 8, 11), to assess their
quantitative relationship.
| |
MATERIALS AND METHODS |
Patient population.
The study group consisted of 59 randomly
selected HIV-1-seropositive persons on various drug protocols from the
outpatient population of the National Institute of Allergy and
Infectious Diseases HIV clinic. A total of 318 samples from these
patients were analyzed.
Informed consent.
Informed consent was obtained from all
patients. Human experimentation guidelines of the U.S. Department of
Health and Human Services were followed in the conduct of the clinical research.
Sample collection and processing. (i) v3 bDNA.
Whole,
EDTA-preserved blood was separated within 2 h of collection, and
plasma was frozen immediately at 70°C in 1-ml aliquots. The samples
were then thawed, spun at 23,000 × g for 60 min at 4°C, and aspirated without disturbing the virus pellet. Pellets were
then frozen at 70°C for no more than 2 days until they could be
tested with the bDNA assay (Bayer Corporation, Diagnostics Division,
Norwood, Mass.).
(ii) RT-PCR.
Whole, EDTA-preserved blood was separated
within 2 h of collection, and plasma was frozen immediately at
70°C in 1-ml aliquots. Aliquots were thawed and then spun at
23,000 × g for 75 min at 4°C, and 800 µl was drawn
off without disturbing the pellet. The resulting pellets in 200 µl of
plasma were then frozen at 70°C for no more than 2 days until they
could be tested with the RT-PCR assay (Roche Diagnostics Corporation,
Indianapolis, Ind.).
(iii) usRT-PCR.
Five-hundred-microliter aliquots of
EDTA-preserved plasma were spun at 23,000 × g for 75 min at 4°C and aspirated without disturbing the virus pellet. Pellets
were then frozen at 70°C for no more than 2 days until they could
be tested with the ultrasensitive (usRT-PCR) Amplicor HIV-1 Monitor
assay (Roche Diagnostics Corporation), as described in the
manufacturer's insert.
Quantitation of HIV-RNA. (i) v3 bDNA.
The Chiron 3.0 bDNA
assay was performed according to the manufacturer's instructions
provided with the assay kit.
(ii) RT-PCR.
The pellets in 200 µl of plasma were
resuspended in lysis buffer and tested according to the test procedure
of the Amplicor HIV-1 Monitor test. No further modification of the
procedure was performed.
(iii) usRT-PCR.
The viral pellets were processed according
to the "UltraSensitive specimen preparation" procedure provided
with the assay kit. With the exception of the alternate specimen
preparation procedures, the test procedure remains the same as that of
the Amplicor HIV-1 Monitor test.
Statistical analysis.
Because viral load tends to change in
a logarithmic fashion and because data are evaluated on the basis of
half-log changes, values were transformed into common
(log10) logarithms and expressed as log10v3
bDNA or log10RT-PCR. Linear regression and correlation were
used to determine the relationship between the v3 bDNA and RT-PCR
values. The methods of Bland and Altman (1) were employed to
assess agreement between the values.
| |
RESULTS |
Correlation and regression.
Assay values were transformed to
common logs. In this analysis, log10RT-PCR is taken as the
predictor variable and log10v3 bDNA is taken as the
response variable. The results are highly correlated. Figure
1 shows a plot of log10v3
bDNA versus log10RT-PCR with the fitted regression line
described by the equation y = 0.0915 + 1.0052 · x, with r = 0.98. The estimate of the
intercept is 0.0915, with a standard error of 0.04. This value
differs from the ideal value of 0.0 at the 0.05 level of confidence.
The value of the slope is 1.0052, with a standard error of 0.01. This value is statistically equivalent to an ideal value of 1.0. A 95%
confidence interval for the estimate of the slope is 0.98 to 1.03, a
range that includes 1.0, indicating that the assays are in good
agreement (discussed below).
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|
FIG. 1.
Scatter plot of log10v3 bDNA versus
log10RT-PCR with the line of equality (solid) and the
fitted regression line (hatched). The equation for the fitted
regression line is given in the lower-right-hand corner, and the
correlation coefficient (r) is given in the upper-left-hand
corner. There is good agreement between the two assays.
|
|
Agreement.
Bland and Altman have pointed out that when a
comparison of two clinical measurements of the same criterion is made,
the use of the correlation may be misleading (1). The
correlation coefficient measures the strength of the relationship
between the variables, but does not necessarily measure the agreement
between them. Figure 2 shows the
difference in log10 assay results
(log10RT-PCR log10v3 bDNA) versus the
average of assay results [(log10RT-PCR + log10v3 bDNA)/2]. The plot shows that the differences in
values are fairly homogeneously distributed between 1.96 standard
deviations above and below the mean (0.072) and are in good agreement
(mean difference in log10 copies/ml ± 2 standard
deviations = 0.072 ± 0.371). A one-sample t test
demonstrates that the mean is significantly different from zero
(P < 0.0001). This implies that the
log10RT-PCR values, on average, are higher than the
log10v3 bDNA values. If the difference values were normally
distributed, one would expect to observe approximately 95% of them
between 1.96 standard deviations of the mean. In fact, 95.28% (303 of
318) of the values fell in this range.
View larger version (22K):
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|
FIG. 2.
Difference in log10 assay results versus
average of log10 assay results. The mean difference (solid
line) ± 1.96 standard deviations (dashed lines) is shown. The
plot shows that the difference values are fairly homogeneously
distributed between 2 standard deviations (SD) above and below the mean
(0.072). A total of 95.28% (303 of 318) of the values fall between the
hatched lines, indicating normal distribution.
|
|
The RT-PCR assay used in these analyses includes a precentrifugation
step of 1 ml of plasma before application to the standard Amplicor
Monitor assay and was used in this laboratory before the UltraSensitive
specimen preparation procedure was included with the Amplicor Monitor
kit. In order to address possible concerns that this sensitized RT-PCR
assay might yield results discordant with those obtained by the
UltraSensitive specimen preparation procedure (11), a subset
of 47 samples was reanalyzed by the UltraSensitive specimen preparation
procedure (usRT-PCR). The results showed that, in fact, the sensitized
and usRT-PCR assays were highly correlated (r = 0.98)
and showed good agreement (mean difference in log10
copies/ml ± 2 standard deviations = 0.041 ± 0.176).
The relationship between values obtained by v3 bDNA and usRT-PCR is
given by the following equation: log10v3 bDNA = 0.608 + 1.1214 · log10usRT-PCR. This
represents a 1.048-fold difference between log10usRT-PCR
values and log10v3 bDNA values.
| |
DISCUSSION |
In the current environment of HIV-1 treatments, it is essential to
be able to quickly and accurately assess viral load. There have been a
number of studies comparing the various techniques available for
monitoring HIV RNA (9, 10). These studies all failed to find
significant agreement between assays, and none revealed a numerical way
to compare the results from the different techniques. Because patients
may be monitored with more than one assay over the course of their
treatment, it is useful to find a quantitative relationship between
these assays.
Our comparison of the third-generation bDNA assay and a modification of
the Amplicor Monitor assay indicates that these two assays are highly
correlated. Although RT-PCR values, on average, are larger than the v3
bDNA values, regression analyses indicate that values are normally
distributed, and the two assays show very good agreement.
In conclusion, we were able to determine that, given an RT-PCR value,
it is possible to derive the comparable bDNA value by the formula
log10v3 bDNA = 0.0915 + 1.0052 · log10RT-PCR.
The knowledge that both assays show good agreement overall and have a
reliable mathematical relationship should assist clinicians in their
use of both data sets when monitoring patient virus load.
| |
ACKNOWLEDGMENT |
This project has been funded completely or in part with federal
funds from the National Cancer Institute, National Institutes of
Health, under contract no. NO1-CO-56000.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Virus Isolation
Laboratory, Clinical Services Program, SAICFrederick, NCI-FCRDC,
Frederick, MD 21702. Phone: (301) 846-1910. Fax: (301) 846-6762. E-mail: rdewar{at}nih.gov.
| |
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Journal of Clinical Microbiology, November 1999, p. 3612-3614, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
