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Journal of Clinical Microbiology, February 2000, p. 914-917, Vol. 38, No. 2
Klinik für Gastroenterologie,
Hepatologie und Infektiologie1 and
Institut für Medizinische Mikrobiologie und
Virologie,2 Heinrich-Heine-Universität,
Düsseldorf, Germany
Received 5 March 1999/Returned for modification 25 March
1999/Accepted 1 November 1999
We have comparatively evaluated Quantiplex version 3.0 and version
2.0 on 133 plasma samples and a repetitive dilution series. Version 3.0 yielded higher human immunodeficiency virus RNA values, and the ratio
of version 3.0 results to version 2.0 results decreased from 3.47 below
1,000 copies/ml to 1.97 above 50,000 copies/ml [linear regression, log
(version 3.0) = 0.915 + 0.871 × log (version 2.0);
r2 = 0.952].
Viral load is strongly correlated
with human immunodeficiency virus (HIV) disease progression (10,
11), and in patients receiving antiretroviral therapy, the degree
of viral load reduction is the best prognostic parameter for clinical
benefit and for sustainability of treatment efficacy (6,
13). Suppression of viral load to below 50 copies/ml is necessary
to achieve durable response to treatment and to delay development of
resistance to antiretroviral drugs (2, 7, 14, 15, 17). We
therefore comparatively evaluated two branched-DNA assays with a low
detection limit, which was reduced from 500 copies/ml in Quantiplex
version 2.0 to 50 copies/ml in version 3.0 (3, 8).
Besides limitations of some assay types with certain subtypes of HIV
type 1 (HIV-1) (12), the lack of approved quantitative HIV-1
standards constitutes a drawback for all HIV quantification systems.
Investigations comparing systems of different manufacturers with
clinical samples show significant differences (4, 9, 16) but
have not yet been performed thoroughly with the new-generation systems
that have lower detection limits of 40 to 80 copies/ml. Thus, if assays
from different manufacturers or different generations of the same
system are used, interpretation and direct comparison of results should
be done with caution.
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Comparative Evaluation of Two Branched-DNA Human Immunodeficiency
Virus Type 1 RNA Quantification Assays with Lower Detection Limits of
50 and 500 Copies per Milliliter
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ABSTRACT
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TABLE 1.
Ratio of assay version 3.0 and
2.0 resultsa
One hundred thirty-three samples were taken successively from
HIV-1-infected patients from our outpatient department. One half of the
samples were taken from patients with a viral load of <500 copies/ml
and the other half were taken from patients with higher values in the
most recent prior measurement (Quantiplex 2.0). Blood was collected
with Vacutainer PPT tubes (Becton Dickinson, Heidelberg, Germany),
centrifuged within 30 min at room temperature, and stored at 
70°C
in the same Vacutainer tubes until batch analysis. Plasma specimens as
well as positive and negative controls were prepared in duplicate for
version 2.0 and singly for version 3.0 according to the manufacturer's
instructions. For dilution series, six additional stored plasma samples
with a previously determined viral load of more than 400,000 copies/ml
and fresh frozen plasma from the blood bank were used. Fresh frozen
plasma was initially added to the 1.5- to 3-ml samples to a total
volume of 13 ml each. An aliquot of 6.5 ml was then repeatedly diluted
1:2 for a total number of 12 samples (dilution factors 1 to 2,048). All
dilution samples were aliquoted, frozen at 70°C for at least 1 h, and thawed immediately before testing. Two separate runs were
performed on each dilution series. Computational statistics were
performed with SAS system 6.12 (SAS Institute Inc., Cary, N.C.).
Analysis of agreement was done according to the difference-against-mean method (1). Curves and regression lines were graphically
fitted by Origin 5.0 (Microcal Software Inc., Northampton, Mass.).
Seventy-nine of 133 serum samples (59%) returned virus counts below
the lower detection limit of 500 copies/ml with version 2.0. Fifteen
(19%) of these samples yielded values above 500 copies/ml with the 3.0 assay (mean, 1,504; median, 1,259; range, 505 to 4,599). Of 54 samples
with more than 500 copies/ml in the 2.0 assay, 3 (5.6%) had fewer than
500 copies/ml in the new assay. Of 69 samples that showed 
500
copies/ml with at least one of the tests, 1 yielded equal values in
both assays and only 4 samples yielded lower values with assay version
3.0 than with version 2.0. Regression analysis of 50 serum samples with
all values within the valid range of 500 to 500,000 copies/ml on
log-transformed values returned the following: [log10
(version 3.0)] = 0.915 + {0.871 × [log10
(version 2.0)]} (r2 = 0.952).
Analysis of agreement (Fig. 1) showed
that the difference between both assays was normally distributed
(P = 0.725) around the mean of the difference. The mean
difference of log10 (version 3.0) log10
(version 2.0) was 0.41 (standard deviation, 0.22). However, there was a
clear trend of a decreasing difference with increasing viral load
[log10 (version 3.0)] [log10 (version
2.0)] = 0.786 {0.091 × [log10 (version
2.0)]}; r2 = 0.079, P = 0.0485}. The same slope was observed with the
dilution series ([log10 (version 3.0)] [log10 (version 2.0)] = 0.623 0.108 × [log10 (version 2.0)];
r2 = 0.175, P = 0.003) although the mean of the difference was lower (0.20). There was
a negative difference between version 3.0 and version 2.0 values in one
of the six dilution series that mainly contributed to this lower mean
of difference. Table 1 further illustrates the trend of a decreasing
difference or ratio from lower to higher viral load ranges.
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The variation coefficient (CV), which is only slightly lower in version 3.0 than in version 2.0 (mean CV over two runs, 11.6 versus 16.9%, respectively) does not explain this trend. The CV distribution over viral load and the Gauss fit curve (Fig. 2) illustrate only that there is, if anything, a slightly higher CV on either side of the viral load range in version 3.0.
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Figure 3 explains better the reason for the between-assay discrepancy, as it shows the "dilution normalized" RNA results of each individual dilution series that were calculated by dividing the RNA results by 2,048 (the highest dilution step) and multiplying them by the respective dilution degree of the individual test sample. In an ideal case, all points of one series should show the same value. However, there is an obvious tendency of increasing values with higher dilution in version 3.0, compared to version 2.0. Therefore, version 3.0 does relatively overestimate RNA values in the lower measurement range.
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In conclusion, both assay versions show high linearity, precision, and concordance. On a linear scale, values from version 3.0 are 3.5 times higher in the lower measurement range and 2 times higher in the upper range. From the clinical point of view, viral load changes of less than 0.5 log are not considered to be significant (5), but the between-assay difference should be taken into account if both test versions are used.
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ACKNOWLEDGMENTS |
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We thank S. Würden for his valuable technical and logistic assistance and J. Hemmer and C. Chiwakata for critically reviewing the manuscript.
The study was supported by Bayer Diagnostics (Emeryville, Calif.), the manufacturer of the Quantiplex kits that were evaluated.
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FOOTNOTES |
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* Corresponding author. Present address: Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany. Phone: 49/40/42818-0 or -370. Fax: 49/40/42818-379. E-mail: manegold{at}bni.uni-hamburg.de.
Present address: Bernhard-Nocht-Institut für
Tropenmedizin, 20359 Hamburg, Germany.
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Journal of Clinical Microbiology, February 2000, p. 914-917, Vol. 38, No. 2
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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