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Journal of Clinical Microbiology, July 2000, p. 2665-2669, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Comparison of Two Amplification Technologies for
Detection and Quantitation of Human Immunodeficiency Virus Type 1 RNA
in the Female Genital Tract
James
Bremer,1,*
Marek
Nowicki,2
Suzanne
Beckner,3
Donald
Brambilla,4
Mike
Cronin,5
Steven
Herman,6
Andrea
Kovacs,2 and
Patricia
Reichelderfer7 for the Division
of Aids Treatment Research Initiative 009 Study
Team
Rush Medical College, Chicago, Illinois
606121; University of Southern
California, Los Angeles, California 900332;
Westat, Rockville, Maryland 208503;
New England Research Institutes, Watertown, Massachusetts
024724; Organon Teknika Corporation,
Durham, North Carolina 277125;
Department of Infectious Diseases, Roche Molecular Systems,
Inc., Alameda, California 945016; and
National Institute of Child Health and Human Development,
Bethesda, Maryland 208927
Received 28 January 2000/Returned for modification 3 April
2000/Accepted 9 May 2000
 |
ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) RNA levels in female
genital tract and peripheral blood samples were compared using two
commercial amplification technologies: the Roche AMPLICOR HIV-1 MONITOR
test and either the Organon Teknika nucleic acid sequence-based
amplification (NASBA-QT) assay or the NucliSens assay. Estimates of
HIV-1 RNA copy number were derived from internal kit standards and
analyzed unadjusted and adjusted to a common set of external standards.
We found a discordance rate of approximately 18% between the two
technologies for the detection of HIV-1 in either the genital tract or
peripheral blood samples. Detection discordance was not consistent
among specimens or among women. There were no significant differences
in adjusted or unadjusted estimates of HIV-1 RNA copy number in the
genital tract samples using the AMPLICOR HIV-1 MONITOR test and either
the NASBA-QT assay or the NucliSens assay. In addition, the estimated
HIV-1 RNA copy number in peripheral blood samples did not differ when tested with the NucliSens assay and the AMPLICOR HIV-1 MONITOR test
using kit standards. However, there was a significant difference in
estimated RNA copy number between the NASBA-QT assay and the AMPLICOR
HIV-1 MONITOR test for internal kit standards, which, as we have
previously shown, was eliminated after adjustment with the external
standards. Our results suggest that the Roche and Organon Teknika
assays are equivalent for quantifying HIV-1 RNA in female genital tract
specimens, although variation in detection does exist.
| |
INTRODUCTION |
Monitoring human immunodeficiency
virus type 1 (HIV-1) RNA in genital tract specimens has become of
primary importance with our growing understanding of the issues
surrounding compartmentalization (4, 11). Measuring HIV-1
RNA has been more complicated in genital tract than in peripheral blood
samples. In semen, nonspecific inhibitors have been associated with
loss of signal (4, 5, 7). However, in reconstruction
experiments with seronegative subjects, no equivalent nonspecific
inhibitors were observed for the female genital tract (8).
That study did not rule out the presence of inhibitors in the genital
tracts of HIV-seropositive women that may be lacking in
HIV-seronegative women.
Genital tract variation has been shown to be greater in women (P. S. Reichelderfer, R. W. Coombs, D. Wright, D. Burns, and A. Kovacs
for the WHS 001 Study Group, Abstr. 38th Intersci. Conf. Antimicrob.
Agents Chemother., abstr. I-251, 1998) than in men (4).
Total variation is a composite of biologic, assay, and sampling
variation. Sampling methods used for the female genital tract
contribute strongly to variation (Reichelderfer et al., 38th ICAAC).
Interlaboratory and interassay variation in patient or spiked samples
of peripheral blood could be significantly reduced by using a common
set of standards (3, 14). However, variation may be
increased in different matrices, and the findings in peripheral blood
samples may therefore differ from those in genital tract samples.
There has been no direct comparison of differences in HIV-1 RNA
levels in the female genital tract among test kits that use the same
sampling method. The objective of this study was to assess the
differences in estimated HIV-1 RNA levels obtained with two commonly
used gene amplification technologies in genital tract samples collected
using the same sampling method.
| |
MATERIALS AND METHODS |
Study population.
This cross-sectional study involved 338 HIV-1-seropositive women enrolled in a longitudinal epidemiologic
cohort study, the Women's Interagency HIV Study (1).
Genital tract specimens and peripheral blood were obtained during one
regularly scheduled 6-month visit between January 1997 and July 1998. The study was limited to women who were not pregnant, who either had
not received therapy or had been on stable antiretroviral therapy for
at least 2 months, and who were not experiencing an active
opportunistic infection. Approximately 40% of the 338 women were on no
therapy, while 60% were receiving combination therapy with or without
a protease inhibitor.
Specimen collection.
Peripheral blood plasma specimens were
collected in acid dextrose citrate tubes, frozen at 
70°C, and
shipped to a central repository. Female genital tract specimens were
obtained by cervical vaginal lavage (CVL) with 10 ml of normal saline.
Aliquots (1 ml) of lavage material, containing both cells and
supernatant, were frozen at 70°C and distributed as described for
the peripheral blood samples. The frozen samples were subsequently
shipped to two independent laboratories.
Quantitative assays.
For this analysis, 338 samples were
tested with the Roche AMPLICOR HIV-1 MONITOR test (10)
(Roche Diagnostic Corporation, Indianapolis, Ind.), 177 samples were
tested with the Organon Teknika Corporation (OTC) nucleic acid
sequence-based amplification (NASBA-QT) assay (13) (Organon
Teknika Corporation, Durham, N.C.), and 162 samples were tested with
the OTC NucliSens assay (6). Specimens were tested at the
Retrovirology Laboratory of Rush-Presbyterian St. Luke's Medical
Center (Roche AMPLICOR HIV-1 MONITOR assay) and the University of
Southern California Medical Center (OTC NASBA-QT and NucliSens assays).
The laboratories used both the kit standards and standards prepared by
the National Institute of Allergy and Infectious Diseases Virology
Quality Assurance Program (VQA; Chicago, Ill.) (9). VQA copy
standards at 0, 1,500, 15,000, and 150,000 copies/ml were included in
the runs to permit the calculations. The assay methods (6, 10, 13) and the VQA standards (9, 14) have been previously described.
For the Roche AMPLICOR HIV-1 MONITOR assay, 0.2 ml of both plasma and
CVL samples was processed using standard Roche plasma processing
procedures. Extracted samples (50 µl) were amplified in a GeneAmp PCR
System 9600 and detected according to the manufacturer's recommendations. Results were calculated according to the
manufacturer's recommendations. The limit of assay sensitivity
provided by the manufacturer was 400 copies/ml for all specimens.
For the OTC NASBA-QT and NucliSens assays, 0.2 ml of peripheral blood
plasma and 0.8 to 1.0 ml of CVL specimens were processed
in accordance
with the manufacturer's instructions using the silica
gel extraction
methodology (
2). For the NASBA-QT assay, 10-fold-diluted
calibrators were used to increase sensitivity (
12). The
NucliSens
assay was run according to the manufacturer's instructions.
In
accordance with the manufacturer's recommendations, the limits
of
detection for the NASBA-QT assay were 500 copies/ml in blood
samples
and 125 copies/ml in genital tract samples; for the NucliSens
assay,
the limits of detection were 400 copies/ml in blood samples
and 80 copies/ml in genital tract
samples.
Statistical analysis.
Analyses included independent
comparisons of the Roche AMPLICOR HIV-1 MONITOR test with the OTC
NASBA-QT and NucliSens assays, as well as a composite comparison of the
AMPLICOR MONITOR and NASBA-QT and NucliSens assays. Comparisons between
assays were based on samples for which estimates were above the limit
of detection.
The analyses reported here assessed the differences among the assays
and the extent to which a common set of standards reduced
the
differences among the assays. Therefore, the results obtained
with the
assays were compared both before and after adjustment
to the VQA
standards. All assays used internal standards; therefore,
adjustment
was made using regressions of estimated RNA concentration
on nominal
log
10 concentration for the VQA standard
estimates.
Comparisons included nominal copy numbers that were above the limits of
detection for the assays under study. The number of
specimens falling
outside the dynamic range of the assay was noted
for each assay system.
The mean, median, and number of values
above the assay cutoff were
calculated for the population as a
whole for each
assay.
The analysis involved pairwise comparisons among assays rather than
three-way comparisons. Paired
t tests were used to test
the
null hypothesis that the average difference in estimated RNA
concentration between assays is 0. Linear regression analysis
was used
to illustrate the effect of sample concentration of HIV-1
RNA on the
correlations among assays and to assess the overall
effect of the
external standard versus the kit standard on HIV-1
RNA copy number
estimates.
| |
RESULTS |
Qualitative differences among assays.
The number of samples
testing positive on each assay is given in Table
1. In peripheral blood samples, there
were 250 samples above the lower limit of detection for the
AMPLICOR HIV-1 MONITOR test, 122 for the NASBA-QT assay, and 96 for the NucliSens assay, for a total of 218 samples above the lower
limit of detection by one or the other OTC assay. For genital tract
specimens, there were 77 samples above the lower limit of detection for
the AMPLICOR MONITOR test, 48 for the NASBA-QT assay, and 32 for the
NucliSens assay, for a total of 80 samples above the lower limit of
detection by one or the other OTC assay. The levels of discordance
between the Roche and OTC assays were 18.6% (63 women) for the genital tract samples and 17.7% (60 women) for peripheral blood samples. For
the 60 discordant blood samples, the RNA values ranged from 402 to
95,000 copies/ml (mean = 4,017). For the 63 discordant CVL
samples, the RNA values ranged from 98 to 100,000 copies/ml (mean = 5,395). Only six of the discordants could be explained by OTC values
in CVL samples below the limit of detection of the Roche assay (400 copies/ml). There was no obvious bias to discordance based on
antiretroviral therapy. Of the 60 and 63 samples discordant in blood
and CVL, 23 and 44%, respectively, were from women not on therapy.
Discordance between assays was not consistent between the two
compartments. Of the 60 and 63 women who were discordant in peripheral
blood and the genital tract, respectively, only 9 were discordant in
both compartments. There was no pattern to the discordance between
assays for these nine women. Four were positive in both compartments by
OTC and negative in both compartments by Roche; two were positive in
both compartments by Roche and negative by OTC; two were positive in
blood by OTC and positive in CVL only by Roche; and one was positive in
CVL by OTC and positive in blood by Roche. Of these nine discordant
samples, only two could be explained by OTC values in CVL samples below
the limit of detection of the Roche assay (400 copies/ml). These two
women were on antiretroviral therapy.
Quantitative differences among assays.
Table
2 shows the descriptive statistics for
both the kit-based and VQA-based estimates of HIV-1 RNA copy number. In
general, HIV-1 RNA copy number estimates were lower for the AMPLICOR
HIV-1 MONITOR test than for either the NASBA-QT or NucliSens assay; this difference was more pronounced in peripheral blood specimens. Adjustment with the VQA standards further lowered the estimates for
both methods and both compartments; this was especially true for
genital tract RNA estimates obtained with the AMPLICOR MONITOR test.
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TABLE 2.
Descriptive statistics for all assays with kit-based and
VQA-based HIV-1 log10 RNA copy number estimates
|
|
Table
3 provides summary statistics for
absolute RNA copy number for specimens above the lower limit of
detection for all
assays. A positive median indicates that values from
the first
assay in the comparison were, on average, higher than values
for
the second assay. The
P values are the results of tests
of the
null hypothesis that the average difference between assays is
0. Kit-based estimates of HIV-1 RNA copy number in peripheral
blood from
the AMPLICOR MONITOR and NASBA-QT assays were significantly
different
(
P = 0.005), but the estimates adjusted to the VQA
standards
were not (
P = 0.96). There was no significant
difference between
the AMPLICOR MONITOR and NucliSens assays for either
kit-based
or VQA-based estimates (
P = 0.42 and 0.28, respectively). In contrast
to peripheral blood, estimates of HIV-1 RNA
copy number in genital
tract specimens were not significantly different
for any assay
method using any set of standards for calculation.
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|
TABLE 3.
Differences in log10 HIV-1 RNA copy number
estimates for all assays for kit-based and VQA-based estimates
|
|
For the AMPLICOR MONITOR and NucliSens assays, the standard deviations
(SD) were 0.16 and 0.31 log
10 HIV-1 RNA copies per
ml,
respectively, based on the performance of the 1,500 VQA copy
number
standard. For the NASBA-QT assay, the SD was 0.30 log
10 HIV-1 RNA copies per ml based on the 15,000 VQA copy number standard.
Thus, the 95% confidence intervals were fairly broad (±0.8
log
10).
Virtually all samples, from both genital tract and
peripheral
blood, fell within ±2 SD of the assays (data not
shown).
Effect of HIV-1 RNA level on assay correlation.
Figure
1 illustrates the linear regression
analysis of the OTC and Roche assays for kit- and VQA-adjusted values
in peripheral blood and genital tract samples. All estimates were
highly correlated (P 
0.001). For genital tract, the
coefficients of correlation between the Roche AMPLICOR HIV-1 MONITOR
test and the OTC NASBA-QT and NucliSens assays were 0.71 and 0.72, respectively, for kit-based determinations and 0.75 and 0.53, respectively, for VQA-based estimates. Similarly, for peripheral blood,
the coefficients of correlation between the AMPLICOR MONITOR test and
the NASBA-QT and NucliSens assays were 0.78 and 0.80, respectively, for
kit-based determinations and 0.59 and 0.74, respectively, for VQA-based estimates. Differences in HIV-1 RNA copy numbers between the
NASBA-QT and NucliSens assays relative to the AMPLICOR MONITOR test
appear to be greater at the ends of the HIV-1 RNA concentration scales, and these differences were not reduced by adjustment with the external
standards.
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FIG. 1.
Linear regression analysis of the OTC and Roche assays
for kit (a and c)- and VQA (b and d)-adjusted values for peripheral
blood (a and b) and genital tract (c and d). Log10 RNA
values from the Roche assay are plotted against those of the NASBA-QT
( ) and NucliSens ( ) assays. Linear regression correlation for the
NASBA and NucliSens assays ( ) is shown, compared to a slope of 1 (---).
|
|
Figure
2 demonstrates the effect of VQA
adjustment of any assay compared to the kit-determined HIV-1 RNA copy
number. For
all assays and both specimen types, the VQA-adjusted copy
number
estimates were higher at higher concentrations and lower at
lower
concentrations. This pattern was most noticeable for the NASBA-QT
assay and peripheral blood specimens. In both peripheral blood
and
genital tract specimens, the effect of VQA adjustment on copy
number
estimates from the NucliSens assay more closely paralleled
the effect
observed for the AMPLICOR MONITOR assay than that observed
for the
NASBA-QT assay.
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[in a new window]
|
FIG. 2.
Effect of VQA adjustment compared to kit-determined
HIV-1 copy number estimates for peripheral blood (a) and genital tract
(b). Log10 RNA values for kit copies are plotted against
VQA-adjusted copy numbers for the Roche ( and ), NASBA-QT ( and ---), and NucliSens ( and
----) assays.
|
|
| |
DISCUSSION |
The results of this study suggest that different gene
amplification methods can be used to arrive at comparable
determinations of HIV-1 RNA copy number in the female genital tract. In
previous studies of blood plasma (3), we observed
differences in estimated RNA copy number between the OTC NASBA-QT assay
and the Roche AMPLICOR HIV-1 MONITOR assay; these differences could be
eliminated by adjusting to a common set of external standards. In the
current study, we confirmed our previous findings concerning the
NASBA-QT and AMPLICOR MONITOR assays but found no significant
differences between HIV-1 RNA copy number estimates obtained with
the NucliSens and AMPLICOR MONITOR assays.
Importantly, there were no significant differences among the assays in
terms of HIV-1 RNA quantification for the female genital tract. Thus,
the use of a common set of external standards would not appear to be
necessary when making comparisons between assays using genital tract
specimens. The absence of a difference between assays using peripheral
blood specimens and those using genital tract specimens could not be
explained by a decrease in the dynamic range. The adjusted and
unadjusted dynamic ranges for peripheral blood and genital tract HIV-1
RNA were equivalent. Similarly, although the assays use different
processing technologies, each uses the same processing technology for
peripheral blood and genital tract specimens.
In contrast to previous findings (3, 4), however, this study
found a higher SD for the OTC assays than for the Roche assay. Thus,
the lack of differences between the estimated RNA copy numbers obtained
with these assays may be confounded by the higher assay SD.
Nonetheless, as previously reported (3), we observed similar
differences when the NASBA-QT and AMPLICOR MONITOR assays were used to
test peripheral blood, in spite of the higher SD.
The effect of VQA standard adjustment was greatest for the
NASBA-QT assay and was more pronounced at higher and lower HIV-1 RNA
concentrations. For both peripheral blood and genital tract specimens
and for both amplification technologies, the copy number estimates
obtained with the VQA standards were higher than the kit-based
estimates at the high end and lower than the kit-based estimates at the
low end.
An issue of concern is the high level of discordance in HIV-1 detection
between the OTC and Roche gene amplification systems. This discordance
was not confined to either compartment or to any group of women. Many
of these differences were not at the level of the limit of detection
for the assays. Sequence variation among the highly discordant isolates
may have resulted in differences in primer pair efficiencies, which
could explain the discordance.
In summary, the OTC NucliSens and Roche AMPLICOR HIV-1 MONITOR assays
appear to be equivalent for determining HIV-1 RNA levels in either the
female genital tract or peripheral blood, although the SD for the
NucliSens assay was high. These equivalent determinations can be made
without using an external set of common standards. However, the two
amplification technologies do produce some discordant positive and
negative results.
| |
ACKNOWLEDGMENTS |
We acknowledge the following individuals for their assistance
with this study: Vinita Goveia, Shirley Lewis, Cheryl Jennings, John
Nelson, Anna Soloviov, and the women enrolled in DATRI 009.
The data in this paper were collected by the Women's Interagency HIV
Study Collaborative Study Group, with centers (principal investigators)
at the New York City/Bronx Consortium (Kathryn Anastos); Brooklyn, N.Y.
(Howard Minkoff); the Washington, D.C., Metropolitan Consortium (Mary
Young); the Connie Wofsy Study Consortium of Northern California (Ruth
Greenblatt and Herminia Palacio); the Los Angeles County/Southern
California Consortium (Alexandra Levine); the Chicago Consortium
(Mardge Cohen); and the Data Coordinating Center (Alvaro Muñoz
and Stephen J. Gange).
This study was supported by the Division of AIDS Treatment Research
Initiative, National Institute of Allergy and Infectious Diseases
(NIAID), National Institutes of Health, Bethesda, Md. (contract no.
AI-15123); the Program Support Center, U.S. Department of Health and
Human Services (contract 282-97-0015, task order 21); and the Women's
Interagency HIV Study, funded by NIAID, with supplemental funding from
the National Cancer Institute, the National Institute of Child Health
and Human Development (NICHD), the National Institute on Drug Abuse,
the National Institute of Dental Research, the Agency for Health Care
Policy and Research, and the Centers for Disease Control and Prevention
(UO1-AI-35004, UO1-AI-31834, UO1-AI-34994, AI-34989, UO1-HD-32632
[NICHD], UO1-AI-34993, and UO1-AI-42590).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Rush Medical
College, Chicago, IL 60612. Phone: (312) 942-3308. Fax: (312) 942-6787. E-mail: jbremer{at}rush.edu.
| |
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Journal of Clinical Microbiology, July 2000, p. 2665-2669, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
