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Journal of Clinical Microbiology, January 2000, p. 85-89, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Relationship of Incremental Specimen Volumes and
Enhanced Detection of Human Immunodeficiency Virus Type 1 RNA with
Nucleic Acid Amplification Technology
Donald J.
Witt,1,*
M.
Kemper,2
Andrew
Stead,1
Christine C.
Ginocchio,3 and
Angela
M.
Caliendo4,
Organon Teknika Corporation, Durham, North
Carolina1; Sacramento Medical
Foundation, Sacramento, California2;
North Shore University Hospital-New York University School of
Medicine, Manhasset, New York3; and
Massachusetts General Hospital, Boston,
Massachusetts4
Received 16 June 1999/Returned for modification 18 August
1999/Accepted 27 September 1999
 |
ABSTRACT |
The relationship between specimen input volume and the frequency of
reported human immunodeficiency virus type 1 (HIV-1) RNA copy numbers
by nucleic acid amplification technology (the NASBA HIV-1 RNA QT
system) was investigated. Results obtained with both clinical specimens
and dilution panels indicated that both the absolute number of reported
results and the reported HIV-1 RNA copy number were directly
proportional to the specimen input volumes evaluated (0.1, 0.5, and 1.0 ml). Conversion of the reported HIV-1 RNA copy numbers to a constant
1.0-ml volume indicated that the numerical relationship among the
specimen input volumes and the HIV-1 RNA copy numbers was
multiplicative. The HIV-1 RNA copy numbers reported for the 0.5-ml
input volume were approximately 5-fold increased over those reported
for the 0.1-ml input volume, and those reported for the 1.0-ml input
volume were 10-fold increased over those reported for the 0.1-ml input
volume. For the specimen input volumes investigated, a common linear
range of 264 to 5,400,000 HIV-1 RNA copies was observed. The use of
increased specimen input volumes did not result in a loss of assay
specificity, as the results reported for specimens from 50 seronegative
blood donors were negative at all three specimen input volumes. In
conclusion, an increase in the input volume of specimens analyzed by
nucleic acid amplification technology can be useful for the enhanced
detection of HIV-1 RNA.
| |
INTRODUCTION |
The sensitivity of nucleic acid
amplification assays to detect and report the analyte target is an
important performance characteristic that has a significant impact on
clinical utility. In the case of human immunodeficiency virus type 1 (HIV-1), assay sensitivity is particularly relevant, as the use of
highly active antiretroviral therapy (HAART) can reduce the amount of
RNA in plasma to levels that are often lower than the detection
capability of a given assay (2, 8).
Approaches to enhancing the sensitivity of nucleic acid amplification
assays for detection of HIV-1 RNA have been directed toward
concentration either of the virion contained in the specimen, by
ultracentrifugation (7), or of the isolated analyte (F. Simons, M. Sjeps, C. De Laat, M. Cronin, and H. Cuypers, Abstr. 6th
Conf. Retrovir. Opportun. Infect., poster 148, 1999). These procedures
are followed by standard nucleic acid amplification techniques for
reverse transcription-PCR (RT-PCR) and nucleic acid sequence-based
amplification (NASBA), respectively. While such approaches may have
efficacy, they require specialized equipment and additional technical
steps that may not be reproducible or efficient in terms of time.
In this study, we conducted experimentation to ascertain the
feasibility of another approach to enhancing the sensitivity of a
nucleic acid amplification assay with only a minor modification of the
standard isolation methodology used with NASBA technology. We reasoned
that the capacity for capture of the HIV-1 RNA analyte on silica
particles by following the methodology of Boom et al. (1)
would be independent of specimen volume. Thus, we sought to determine
whether an increase in the size of the specimen volume used for
analysis resulted in greater clinical sensitivity, which is defined in
terms of the number of specimens with reported HIV-1 RNA copy results,
without loss of specificity. Further, the mathematical relationship
between the reported HIV-1 RNA copy number and the volume of the
specimen input was determined.
| |
MATERIALS AND METHODS |
Nucleic acid amplification assay.
The NASBA HIV-1 RNA QT
system (Organon Teknika Corp., Durham, N.C.) was used as the nucleic
acid amplification assay per the manufacturer's directions
(9). The assay incorporates a stringent nucleic acid
isolation procedure based on the release of RNA with detergent and
guanidine thiocyanate followed by capture on silica particles
(1). The numerical ratio of the specimen input volume to the
lysis buffer volume was held constant for the three specimen input
volumes studied: 0.1 ml of the specimen and 0.9 ml of NASBA lysis
buffer, 0.5 ml of the specimen and 4.5 ml of NASBA lysis buffer, and
1.0 ml of the specimen and 9.0 ml of NASBA lysis buffer. Following the
addition of the specimen to the different volumes of NASBA lysis
buffer, the specimen was mixed thoroughly by rocking. The subsequent
steps of the assay (i.e., isolation, amplification, hybridization, and
detection) were performed identically for all specimen input volumes as
provided by the manufacturer.
Clinical specimens.
Specimens were collected from
HIV-1-infected patients from the North Shore University Hospital Center
for AIDS Research and Treatment (Manhasset, N.Y.) and from the Miriam
Hospital (Providence, R.I.) over a 4-month period from July to November
1996. Demographic information concerning the patients' overall health
status and medication was obtained from each of the subjects. No
exclusionary criteria were established for subject participation in the
study. Informed consent was obtained from each subject prior to
specimen collection. Peripheral blood was collected by venipuncture
from each subject by using EDTA-VACUTAINER tubes (Becton Dickinson and
Co., Franklin Lakes, N.J.). Each specimen was processed by standard
techniques to obtain plasma, which was used for the subsequent nucleic
acid amplification testing. Aliquots of the plasma specimens were added
to each of the three volumes of lysis buffer at the volumes indicated
above and were stored at 
70°C until they were tested. Previous
studies performed with the NASBA HIV-1 RNA QT system have indicated
that no observable loss of reported HIV-1 RNA occurs following thawing
after ultralow-temperature storage in lysis buffer (6). Each
specimen was tested three times with the NASBA HIV-1 RNA QT assay, once
with each of the three specimen input volumes.
Blood donor specimens.
Whole blood was collected from 50 volunteer donors into EDTA-VACUTAINER tubes. Plasma was obtained by
standard techniques. Aliquots of the plasma specimens were added to
each of the three volumes of lysis buffer as described above.
Dilution studies. (i) Terminal dilution of clinical
specimens.
Ten clinical specimens containing HIV-1 RNA were
diluted in lysis buffer to the end point for the 0.1-ml specimen input
volume. Additional aliquots of the same specimens at input volumes of 0.5 and 1.0 ml were also tested at each dilution in order to determine the relationship between the specimen input volume and detectability by
the NASBA HIV-1 RNA QT system at decreasing concentrations of the
analyte. Each specimen of the dilution series was tested three times at
each input volume, for a total of nine tests for each dilution. The
total number of tests performed was 360.
(ii) Terminal dilution of in vitro culture stock.
Studies
were performed with a set of serially diluted specimens derived from
HIV-1-infected cell cultures; the virus-infected cell cultures
originated from three HIV-1 clinical isolates (11). The
concentration of HIV-1 virions from the supernatant of the culture was
estimated by electron microscopy and p24 determination at 5 × 108 HIV-1 RNA copies/ml. The stock material was diluted in
twofold steps by using a fresh-frozen plasma unit (600 ml containing 55 ml of anticoagulant sodium citrate solution, USP) from a seronegative blood donor as the diluent to create a series of 16 specimens with
different concentrations of HIV-1 RNA. The estimated concentrations of
HIV-1 RNA in the dilution series ranged from 5.4 × 106 to 1.65 × 102 copies/ml in the
dilution series. Each specimen at each dilution was tested six times
for each input volume, for a total of 288 tests.
CD4+ cell concentration determination.
CD4+ lymphocyte concentrations in the clinical specimens
were determined by standard flow cytometry techniques.
Statistical analysis.
The linear ranges for each specimen
input volume of the in vitro culture stock dilutions tested with the
NASBA HIV-1 RNA QT system were determined by using linear regression
models. The HIV-1 RNA copy number reported for each specimen was
regressed on the expected number of input copies, as provided by the
AIDS Clinical Trial Group Viral Quality Assurance Laboratory
(Rush-Presbyterian-St. Luke's Medical Center, Chicago, Ill.). Since
variability in copy number upon repeated testing of the same specimen
is directly proportional to the mean, both dependent and independent
variables were logarithmically transformed prior to analysis. After
transformation, the residual variation in the dependent variable at
each level of the independent variable was assumed to be Gaussian with
zero mean and constant variance.
Separate data sets were formed from the data for each specimen volume
input level, and consequently each data set represented a different
range of the x variable. For each data set, a test of
linearity was performed by partitioning regression analysis residual
variation into two components: one representing error due to lack of
fit to a linear model and the other representing pure error
(3). The ratio of expected mean squares of these sources of
error follows an F distribution. For each range, an F value estimating this ratio was computed, and the
probability (P) of obtaining an F value greater
than the observed value under a null hypothesis of no lack of fit to a
linear model was calculated. A P value lower than 0.15 was
interpreted as evidence of nonlinearity.
| |
RESULTS |
Clinical specimens from HIV-1-infected individuals.
Of the 102 specimens evaluated with the three investigational input volumes, HIV-1
RNA was reported by the NASBA HIV-1 RNA QT system for the majority (66 of 102; 65%) with all three input volumes. For 15 specimens (15%), no
HIV-1 RNA was reported by the NASBA HIV-1 RNA QT system with any of the
three input volumes tested; of these, 14 (93%) were from patients
receiving antiretroviral therapy. For another 13 specimens (13%),
HIV-1 RNA was reported by the NASBA HIV-1 RNA QT system with two input
volumes, 0.5 and 1.0 ml. For eight specimens (8%), HIV-1 RNA was
reported by the NASBA HIV-1 RNA QT system only with the highest input
volume, 1.0 ml. The numbers of specimens for which HIV-1 RNA was
reported were similar among the three groups stratified by
CD4+ counts for each of the input volumes (Table
1).
The rate of HIV-1 RNA copy number reporting with the NASBA HIV-1 RNA QT
system was proportionately related to the specimen
input volumes when
all specimens were considered cumulatively.
The highest detection rate
for HIV-1 RNA was found with the 1.0-ml
specimen input volume (87 specimens; 85%). The next greatest number
of specimens for which HIV-1
RNA was reported was obtained with
the 0.5-ml input volume (79 specimens; 77%). A specimen input
volume of 0.1 ml resulted in the
lowest rate of HIV-1 RNA detection
(66 specimens; 65%). These results
indicated a significant increase
with the use of larger specimen input
volumes compared to the
0.1-ml volume (a 15% increase with the 0.5-ml
input volume and
a 21% increase with the 1.0-ml input
volume).
The HIV-1 RNA copy number reported by the NASBA assay was also
consistent as a function of specimen input volume. For each
clinical
specimen tested, the highest HIV-1 RNA copy number was
reported with
the 1.0-ml input volume (mean = 169,430) and the
lowest copy
number was reported with the 0.1-ml input volume (mean
= 19,909).
The HIV-1 RNA copy numbers reported with the 0.5-ml
input volumes were
intermediate between those with the 0.1-ml
input volume and those with
the 1.0-ml input volume (mean = 85,299)
for each clinical
specimen. For the clinical specimens tested,
the HIV-1 RNA copy numbers
observed were in relatively good agreement
with the HIV-1 RNA copy
numbers expected based on the interval
relationship of the three
specimen input volumes. The mean HIV-1
RNA copy number for the 0.5-ml
input volume was 4.3 times that
for the 0.1-ml input volume, and that
for the 1.0-ml input volume
was 8.5 times that for the 0.1-ml input
volume. The mean HIV-1
RNA copy number for the 0.5-ml input volume was
50% of the mean
HIV-1 RNA copy number obtained with the 1.0-ml input
volume. In
no case was HIV-1 RNA reported with the smallest specimen
input
volume and not reported with the two larger input
volumes.
Whole-blood donor specimens.
To determine whether an increase
in specimen input volume might affect the specificity of the assay,
specimens from 50 HIV-1-seronegative volunteer whole-blood donors were
evaluated with each of the three input volumes. No HIV-1 RNA copies
were reported from any of the 50 specimens evaluated at any of the
three input volumes.
Dilution studies.
Dilutions of both clinical specimens from
HIV-1-infected individuals and a characterized stock were analyzed to
determine the relative sensitivities of the three input volumes. This
empirical approach with the stock material also enabled estimation of
the linear range for each specimen input volume. For each of 10 clinical specimens diluted to the end point of detection by the assay, the reporting of HIV-1 RNA reached the assay threshold first with the
0.1-ml input volume. Further dilutions were necessary to reach the end
point for detection with both the 0.5- and the 1.0-ml input volume. The
relationship between the dilution required to attain the end point and
the input volume was directly proportional, as exemplified in Fig.
1. The mean HIV-1 RNA copy number
reported at each dilution was proportional to the input volume (Table
2). As with the undiluted clinical
specimens tested with different input volumes, there was no specimen
for which HIV-1 RNA was reported at the 0.1-ml input volume and not at
the larger input volumes.
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FIG. 1.
Relationship of specimen input volume to HIV-1 RNA copy
number reported by the NASBA HIV-1 RNA QT system for a representative
clinical specimen that was terminally diluted.
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TABLE 2.
Relationship between reported HIV-1 RNA copy numbers in
terminally diluted clinical specimens and specimen
input volume
|
|
Detection of HIV-1 RNA following dilution of the in vitro HIV-1 stock
followed the same general pattern as that observed for
the clinical
specimens. For the stock material, the threshold
of detection by the
NASBA HIV-1 RNA QT system was attained first
in the dilution series
with the 0.1-ml specimen input volume and
last with the 1.0-ml specimen
input volume. The threshold of detection
for specimens with the 0.5-ml
input volume was at dilutions intermediate
between those with the
highest and lowest input volumes (Table
3). Subsequent conversion of these data
to a constant 1.0-ml
input volume and logarithmic transformation of the
data indicated
no detectable difference in the reported number of HIV-1
RNA copies
as a function of input volume (Figures
2A and
B). The reported
HIV-1 copy numbers were
found to be described as a linear function
across a defined range of
HIV-1 RNA copy numbers from 264 to 5,400,000
for all three specimen
input volumes, and no evidence on a log-log
scale of a departure from
linearity was observed (
P = 0.44). In
this linear range
of HIV-1 RNA copy numbers, there was no significant
difference between
the intercepts (
P = 0.44) or slopes (
P = 0.57)
describing the results with the three specimen input
volumes.
As a result, a common slope and intercept could be used to
describe
the relationship [log
y = 0.419 + 0.914 · log
x] (Fig.
2C). Thus,
these results indicate
that the number of copies reported was
directly proportional to the
number of copies expected per milliliter
multiplied by the specimen
input volume.
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|
TABLE 3.
Relationship of reported HIV-1 RNA copy numbers in a
terminally diluted HIV-1 stock and specimen
input volumes
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|
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FIG. 2.
Number of HIV-1 RNA copies observed with the NASBA HIV-1
RNA QT system versus the number of copies expected based on an
estimated input of 1 ml (A) or adjusted for specimen input volume (B).
(C) Observed linear range. The input specimens were derived by dilution
of a well-characterized HIV-1-infected cell lysate provided by the
Viral Quality Assurance Laboratory.
|
|
| |
DISCUSSION |
The results of the present study demonstrated a constant
proportional relationship between the specimen input volume and the number of HIV-1 RNA copies reported following amplification with the
NASBA HIV-1 RNA QT system. This relationship was observed both with
authentic clinical specimens from HIV-1-infected individuals and with
specimens derived from an in vitro HIV-1 stock. Conversion of the
latter data to reflect input on a copy number basis indicated no
statistical difference between the three specimen volumes with respect
to the relationship of input copy to copy number of reported HIV-1 RNA.
The consistently observed relationship between specimen input volume
and HIV-1 RNA copy number should be extrapolatable to other specimen
input volumes if the reported HIV-1 RNA copy number is within the
defined linear range of the assay (264 to 5,400,000 copies). This
extrapolation was reported in a previous study using a modified version
of the NASBA HIV-1 RNA QT assay with specimen input volumes of 0.2 and
1.0 ml (5). In addition, by increasing the volume of the
specimen used for analysis with this assay, an increase in the number
of specimens with HIV-1 RNA copies reported (clinical sensitivity) was achieved.
Efficacious utilization of viral load testing is predicated upon the
ability to obtain an accurate report of the HIV-1 RNA copy number in a
given patient specimen. The ability of a nucleic acid amplification
assay for determination of HIV-1 RNA to report copy numbers accurately
under circumstances where the concentration of HIV-1 RNA is diminished,
for example, following administration of HAART, is especially important
clinically. Clearly, detection of low levels of HIV-1 RNA is essential
for clinical management of patients receiving anti-HIV-1 therapeutic
regimens (see, for example, reference 5).
The difficulty of detecting low analyte concentrations with standard
HIV-1 viral load assay configurations has led to the development of
assay refinements to improve detection sensitivity. Concentration of
the isolated HIV-1 RNA or of virions from the specimen forms the
conceptual basis for such approaches. In the present study, our
approach utilized the flexibility of the Boom nucleic acid isolation
procedure to increase the specimen input volume, with a concomitant
proportional increase in the NASBA lysis buffer volume, and thereby
increase the concentration of the HIV-1 RNA analyte available for
amplification. In effect, capture of the analyte by the silica matrix
used for the Boom nucleic acid isolation procedure is analogous to
physical concentration of HIV-1 virions by ultracentrifugation
(10). The approach described in this study may be generally
more useful as a means for improving the sensitivity of HIV-1 RNA
detection, since the probability of HIV-1 RNA detection increases with
large specimen volumes. This approach may reduce the necessity of
performing a second ultrasensitive test procedure if the initial viral
load test result is below the detection limit (4). Further,
since the analysis of greater specimen volumes can be accomplished with
a minor modification of the standard NASBA isolation procedure, this
approach may be more efficient in terms of time than the reported
ultrasensitive procedures used in conjunction with HIV-1 RNA viral load testing.
In conclusion, the use of specimen volumes increased as much as 10-fold
over the normal 0.1-ml input for the NASBA HIV-1 RNA QT assay resulted
in an increased frequency of HIV-1 RNA reporting. The HIV-1 RNA copy
numbers reported by the NASBA HIV-1 RNA QT system were found to be
directly proportional to the specimen input volume. The observed
increase in clinical sensitivity can be attributed to the ability of
the Boom nucleic acid isolation procedure to efficiently capture HIV-1
RNA from incremental specimen input volumes.
| |
ACKNOWLEDGMENTS |
Organon Teknika Corporation, the manufacturer of the NASBA test
kits, provided funding for this work. C. C. Ginocchio was funded
in part by the Jane and Dayton Brown and Dayton Brown, Jr., Molecular
Diagnostics Laboratory.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Organon Teknika
Corp., 100 Akzo Ave., Durham, NC 27712. Phone: (919) 620-2392. Fax: (919) 620-2324. E-mail: dwitt{at}orgtek.com.
Present address: Emory University Hospital, Atlanta, Ga.
| |
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Journal of Clinical Microbiology, January 2000, p. 85-89, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
