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Previous Article | Next Article 
Journal of Clinical Microbiology, June 2000, p. 2348-2353, Vol. 38, No. 6
University of North Carolina, Chapel Hill,
North Carolina 275991; New England
Research Institute, Watertown, Massachusetts
024722; University of Washington,
Seattle, Washington 981043; Cleveland
Clinic, Cleveland, Ohio 441954;
Rush-Presbyterian Hospital, Chicago, Illinois
606125; University of Southern
California, Los Angeles, California 900336;
Walter Reed Army Hospital, Rockville, Maryland
208527; Children's National Medical
Center, Washington, D.C. 200108; and
National Institute of Child Health and Human Development,
Rockville, Maryland 208929
Received 22 November 1999/Returned for modification 23 February
2000/Accepted 15 March 2000
We have evaluated two commercially available kits (AMPLICOR MONITOR
[Roche] and NASBA HIV-1 QT or NucliSens HIV-1 QT [Organon Teknika])
and two noncommercial methods for the accurate quantitation of human
immunodeficiency virus type 1 (HIV-1) RNA in seminal plasma. The same
panels of coded specimens were tested on four separate occasions.
Laboratories using the commercial assays employed silica beads to
isolate HIV-1 RNA, which removed inhibitory factors sometimes found in
seminal plasma. Sensitivities and specificities, respectively, for each
assay were as follows: AMPLICOR MONITOR, 100 and 73%; NASBA HIV-1 QT,
84 and 100%; NucliSens HIV-1 QT, 99 and 98%; and noncommercial
assays, 91 and 73%. When results from the laboratory that was
inexperienced with the silica bead extraction method were excluded from
the analysis, specificity for the Roche assay increased to 100%. The
commercial assays demonstrated highly reproducible results, with
intra-assay standard deviations (measured in log10 RNA
copies/milliliter of seminal plasma) ranging from 0.11 to 0.32; those
of the noncommercial assays ranged from 0.12 to 0.75. Differences in
mean estimated HIV-1 RNA concentrations were Sexual transmission of human
immunodeficiency virus type 1 (HIV-1) is postulated to occur through
direct exposure of the oral, vaginal, urethral, or rectal mucosal
surfaces to genital secretions (semen and cervical vaginal secretions)
from a sexual partner. In 1996 the Centers for Disease Control and
Prevention indicated that sexual transmission of HIV-1 was the primary
route of HIV-1 infection in the United States in persons aged 25 to 44 years (3). If blood and semen HIV-1 concentrations are
predictive of the probability of HIV-1 transmission, then strategies to
decrease the viral load below a critical level in both these
compartments may ultimately reduce the rate of transmission.
Recovery of infectious HIV-1 from seminal cells has been highly
variable (9 to 55%) and also inconsistent within an individual over
time in longitudinal studies (11). Seminal plasma
infrequently yields infectious virus (10, 18). Early efforts
to quantitate virus in seminal plasma used noncommercial reverse
transcription (RT)-PCR amplification techniques (1, 9, 12, 14,
17) and employed external standards as the method of
quantification. When RT-PCR kits that used internal quantitation
standards became commercially available, it became clear that seminal
plasma often contained inhibitors of the RT-PCR. This inhibition could
be removed by using silica beads to isolate the viral RNA (5, 6,
8).
Multicenter clinical drug trials are now actively studying the
effects of different antiretroviral drug regimens on the viral load in
blood and the genital tract, including the evaluation of viral load in
genital secretions in relation to HIV-1 transmission. We have evaluated
the various methods in the processing, isolation, and quantitation of
HIV-1 RNA in seminal plasma and developed a standardized method to
facilitate the comparison of data generated by studies in the field.
This manuscript describes the technical performance characteristics of
both commercial and noncommercial HIV-1 RNA assays which have been used
to measure seminal plasma HIV-1 RNA and compares blood plasma with
seminal plasma as a dilution matrix for the preparation of controls and
proficiency panels.
Quantitative HIV RNA assays.
Two commercially available
assays were compared: the Roche AMPLICOR MONITOR assay and the Organon
Teknika NASBA HIV-1 QT assay or NucliSens HIV-1 QT assay (hereafter in
this work the Organon Teknika assays are referred to as NASBA and
NucliSens, respectively). These assays have been described elsewhere
(7, 13, 16). Additionally, two laboratories employed
noncommercial assays. The Roche MONITOR assay was used according to the
manufacturer's instructions except for HIV-1 RNA isolation. Instead,
viral RNA was isolated using the silica bead procedure of Boom et al.
(2). Previous experiments had demonstrated that factors in
some seminal plasma specimens inhibited RT-PCR amplification and that
these inhibitors were removed by silica bead extraction (5, 6, 8). The limit of detection of the MONITOR assay is 400 copies/ml.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Multicenter Evaluation of Methods To Quantitate
Human Immunodeficiency Virus Type 1 RNA in Seminal Plasma
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.67 log10
and were greater at low viral loads. Suspension matrices that used
blood plasma or seminal plasma did not make a difference in recovery of
HIV-1 RNA, which suggested that blood plasma specimens can be used as
external controls for seminal plasma assays. More variation in the
HIV-1 RNA viral loads was observed in the seminal plasma values than in
the blood plasma values when paired specimens from HIV-1-infected men
were tested. Quantitation of HIV-1 RNA in seminal plasma can be
reliably accomplished using two commercially available assays, and may
be incorporated into the evaluations of HIV-1 seropositive men enrolled
in clinical studies.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
14,000 × g for 60 min at
4°C. Pellets were then resuspended in Tri Reagent (4), and
viral RNA was extracted with chloroform and isopropanol. The HIV-1 RNA was then subjected to RT-PCR using published methods (14,
15). Briefly, HIV RNA was reverse transcribed to cDNA that was
then amplified using Taq polymerase and either SK38 and SK39
primers over 35 cycles (14) or the following primers: Sense,
CAATGAGGAAGCTGCAGAATGGGATAG, and Anti-sense,
CATCCATCCTATTTGTTCCTGAAGG. Amplicons were detected by
hybridization with a 32P-labeled SK19 probe or
ATGAGAGAACCAAGGGGAAGTGACATAGCA (15). The RNA copy
numbers were estimated by comparison to a standard curve.
Participating laboratories. Eight laboratories (coded A to H) participated in the evaluation at one or more times. Technologists performing the respective assays had previous experience in the performance of the nucleic acid amplification assays using blood plasma. However, two laboratories (B and E) did not have previous experience with the Boom silica bead method of viral RNA extraction (2) and four laboratories (B, D, E, and F) did not have previous experience with viral RNA measurements from seminal plasma specimens. In the first round of testing, two laboratories (one experienced in testing seminal plasma [A] and one inexperienced [B]) used the Roche MONITOR assay. Similarly, an experienced laboratory (C) and an inexperienced laboratory (D) used the NASBA assay. Two laboratories used their own noncommercial assay (laboratories G and H). For the second and third rounds of testing, two different laboratories (E and F), neither of which had experience with seminal plasma viral RNA determination, assayed the panels using the Organon Teknika assay, replacing laboratory D. Overall, six laboratories tested seminal plasma panel 1 (SP01), seven tested panel 2a (SP2a), five tested panel 2b (SP02b), and five tested panel 3 (SP03).
Composition of test panels. The Virology Quality Assurance Laboratory of the National Institutes of Health provided three separate blinded panels of specimens for testing (SP01, SP02, and SP03) to each of the participating laboratories. Panels SP01 and SP02 consisted of pooled HIV-negative seminal plasma diluted 1:1 in either Hanks' balanced salt solution (SP01) or phosphate-buffered saline (SP02) into which a known quantity of HIV-1 was added (henceforth referred to as the nominal concentration of HIV-1 RNA). Panel SP01 consisted of six specimens each at nominal concentrations of 103 and 104 HIV RNA copies/ml, five specimens each at 105 and 106 copies/ml, and two negative specimens. Panel SP02 consisted of four negative specimens and five replicates at each of the following nominal concentrations: 103, 104, 105, and 106 HIV RNA copies/ml. The viral stock used was a highly characterized subtype B HIV-1 obtained from pooled culture supernatants from three HIV-1-seropositive donors (19). Virology Quality Assurance Laboratory standards used in each panel were prepared by diluting the same viral stock into HIV-1-seronegative blood plasma. Panel SP02 was tested on two separate occasions approximately 4 months apart, designated SP2a and SP2b. Panel SP03 consisted of triplicate aliquots of paired blood plasma and cell-free seminal plasma from one HIV-1-seronegative and five HIV-1-seropositive donors. Specimens were shipped frozen via overnight express to each of the participating laboratories.
Statistical methods. Estimates of HIV-1 RNA concentration were first transformed to the log10 scale. The intra-assay standard deviation (SD) of log-transformed estimates from each panel in each laboratory was estimated from the mean square error for a one-way analysis of variance, with the nominal concentration as the predictor variable. Estimates that were below the limit of detection were set at the limit for each kit. This negatively biased the SDs, but the bias was confined to a few laboratories and was small, given the small number of values below the limit of detection. Estimates were compared among laboratories using regressions of log10 estimated HIV-1 RNA concentration on log10 nominal HIV-1 RNA concentration. Both slopes and intercepts were allowed to vary among laboratories in the initial model for each panel. If no differences among slopes were detected on a panel, then a model with parallel regression lines was assumed. Under this model, differences among laboratories did not vary with nominal concentration and the differences were estimated from the intercepts for the regressions. However, if slopes did differ, then the differences in estimated RNA concentration among laboratories varied with RNA concentration. When this occurred, the regression lines were compared graphically to determine if the differences increased or decreased with concentration, and to estimate the magnitude of interlaboratory variation. A similar regression approach was used to compare data between rounds of testing on panel SP02, which used different nonplasma dilution matrices, within laboratories and to compare estimates from spiked seminal plasma and blood plasma for this panel.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Sensitivity and specificity of assays used for the quantitation of
HIV-1 RNA from seminal plasma.
Tables
1 and 2
summarize the performance for each assay and participating laboratory
for the first two panels. Six laboratories (A, B, C, D, G, and H)
participated in the first round of testing (SP01) (Table 1). One
laboratory, inexperienced in the Boom RNA extraction method and using a
commercial kit, reported three false-positive results (the two negative
seminal plasma specimens and the negative control), all of which had
high estimates of viral RNA (6,000 to 11,000 copies/ml). The two
laboratories that used the NASBA assay reported false negatives from
samples at 103 viral RNA copies/ml. Five of the six false
negatives from laboratory H occurred at 103 viral RNA
copies/ml, and one occurred at 104 viral RNA copies/ml.
|
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Intra-assay variation.
Standard deviations for log-transformed
estimates from seminal plasma varied widely among panels and assays
(Tables 1 and 2 and data not shown for SP03) (range of
log10 SDs: 0.11 to 0.75 viral RNA copies/ml). The Roche
MONITOR assay was the most consistent, with all log10 SDs
being 0.19 viral RNA copies/ml. Most of the log10 SDs for
the NASBA and NucliSens assays were <0.20 viral RNA copies/ml, but
values of 0.29 and 0.32 were also obtained. The highest SDs were
obtained from noncommercial assays (range of log10 SDs,
0.12 to 0.75 viral RNA copies/ml).
0.15 to 1.59, with 12 of the 14 values between 0.15 and 0.79. In contrast, laboratories
which used noncommercial assays had more shallow slopes (0.59 to 0.89)
and higher intercepts (1.50 to 2.87), indicating that HIV-1 RNA
estimates from these laboratories would tend to be higher than the
estimates obtained in the five laboratories that used the commercially
available kits. In addition, data from the laboratories that used
commercial kits were very precise (r2 0.93), compared with data from the other two laboratories
(r2, 0.36 to 0.98).
Intralaboratory variation.
The consistency of performance
within each of five of the laboratories that used commercial assay kits
was assessed by comparing estimates from the two rounds of testing of
panel SP02 that occurred 4 to 5 months apart for both seminal and blood
plasma (Table 2). Despite the use of different kit lots between runs
within laboratories and the different suspension matrices, the results
were highly reproducible (Fig. 1 [panels
for laboratories A, B, C, E, and F]). Intra-assay variation was
greater for samples with 103 HIV-1 RNA copies/ml than those
with higher concentrations, especially in laboratories that used the
NucliSens assay (Fig. 1 [panels for laboratories C, E, and F]).
Overall there was remarkable consistency in the results observed within
laboratories as well as between laboratories (Fig. [bottom right
panel]).
|
Effect of type of body fluid matrix on the measurement of HIV-1 RNA. We compared estimates of numbers of HIV-1 RNA copies per milliliter obtained from the spiked seminal plasma samples and the spiked blood plasma controls to determine if differences in composition of the suspension matrix affected estimates of HIV-1 RNA concentration (panel SP02). Neither the slopes nor the intercepts differed significantly between spiked seminal plasma samples and spiked blood plasma controls in four of the five laboratories that used the commercial assays (laboratories A, C, E, and F). The fitted regression lines for the semen samples and plasma standards were essentially superimposable, indicating that the estimates from the two were almost identical. The eight regression lines derived from laboratories A, C, E, and F were also extremely precise (r2 > 0.95). RNA recovery from the blood plasma samples was poor in laboratory B, resulting in a flatter slope and higher intercept than those obtained from seminal plasma. However, the great similarity of the paired regression lines in four of the laboratories indicates that the suspension matrix did not make a difference at least when the Boom RNA isolation method was used.
Variation within specimen type.
In panel SP03 paired blood
plasma and seminal plasma from HIV-1-seropositive donors were evaluated
using the two commercial assays. Results were consistent between assays
and among laboratories (Fig. 2). Slightly
more variation in copy number (average 0.05 log10 SD) was
observed when replicates of seminal plasma (Fig. 2B) were tested than
was observed with blood plasma (Fig. 2A). The median SD (measured in
log10 viral RNA copies/milliliter) for blood plasma was
0.08 for the MONITOR assay and 0.06 for the NucliSens assay. In
contrast, the median SD for seminal plasma was 0.12 for the MONITOR
assay and 0.10 for the NucliSens assay. None of these differences were
statistically significant.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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HIV-1 RNA quantitation assays were originally developed to assess viral RNA levels in blood plasma. Some of these assays have also been used, sometimes with modification, to measure viral load in seminal plasma. We evaluated several of the previously published methods for quantifying HIV-1 RNA from seminal plasma (5, 6, 14, 15) and found remarkable consistency in HIV-1 RNA quantification in seminal plasma among laboratories, even for those laboratories that had little prior experience working with semen.
Our data indicate that the commercial assays provide more sensitive and consistent results when used to assess viral load in the seminal plasma than do noncommercial assays. These observations may be a consequence of the internal standards used by both of the commercial assay kits, which can signal the presence of a problem with inhibition of viral nucleic acid amplification. In addition, reagents for commercial kits are prepared in large, well-characterized lots. All laboratories in this study that used a commercial HIV-1 RNA assay kit also used the silica procedure for viral RNA extraction, while those laboratories that used noncommercial assays did not. Previous studies have indicated that seminal plasma frequently contains inhibitors of RT-PCR and that the silica bead isolation extraction procedure (2) can remove these inhibitors (5, 6, 8).
Although all of the laboratories which participated in these evaluations were very experienced in performing HIV RNA assays, laboratories B and E had never used the silica bead RNA isolation procedure and laboratories B, D, E, and F had never worked with seminal plasma. In particular, laboratories A and B prepared their own silica beads and reagents, while laboratories C, D, E, and F used commercially available reagents which were part of the Organon Teknika HIV RNA kits. Laboratory B experienced the most problems, obtaining several false positives in the first two panels tested. By the third round of testing (panel SP02b), more-consistent results were obtained. These results demonstrate that inexperience in just one step in an assay can affect laboratory performance and also indicate the value of quality assurance program evaluations. Laboratories initiating studies that require silica bead extraction techniques might be advised to use the commercially available reagents.
There appeared to be no effect of the suspension matrix on the ability to estimate HIV-1 RNA levels when the Boom silica RNA extraction method and a commercial HIV-1 RNA quantification kit were used. This is an important practical consideration, as it suggests that the controls that were provided by the manufacturer with each assay kit can be reliably used in assays in which seminal plasma is the test specimen. We did observe somewhat more variation when replicates of seminal plasma were tested than when blood plasma was assayed (Fig. 2). The reasons for this are unclear but may be related to the intrinsic complex biological nature of seminal plasma and the fact that it is composed of fluids from several reproductive compartments. Part of the increased variation observed among the seminal plasma samples may also be due to the generally lower copy numbers found in the seminal plasma compared to blood plasma.
In summary, the NucliSens and AMPLICOR MONITOR kits were found to quantify HIV-1 RNA in seminal plasma reliably and comparably after isolation of viral RNA using the silica bead procedure of Boom (2). This suggests that data derived from one of the commercial assays could be easily compared with data from the other, as might occur in different clinical trials or natural history studies, and that data sets could perhaps be pooled to achieve larger numbers and possibly more statistical power for analysis purposes. The accurate and reliable quantification of HIV-1 viral load in the seminal plasma by these methods is fundamental to the evaluation of therapeutic drug and vaccine candidates aimed at the reduction of viral load in the semen and the subsequent reduction in HIV-1 transmission.
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ACKNOWLEDGMENTS |
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We acknowledge the expert technical assistance of Joan Dragavon, Michelle Jack, Reggie Sampoleo, Jody Schock, and Colleen Starkey. We also thank Suzanne Granger for data analysis and graphics. Michael Cronin of Organon Teknika provided NASBA and NucliSens kits used by some of the participating laboratories.
Financial support was provided by the following grants and contracts: AI-27664, AI-30731, and AI27757 (to R.W.C.) and AI-25868 and DK-49381 (to S.A.F.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, CB# 7140, Chapel Hill, NC 27599-7140. Phone: (919) 966-6872. Fax: (919) 966-9873. E-mail: fiscussa{at}med.unc.edu.
Present address: Diagnostic Products Corp., Los Angeles, Calif.
Other members of the AIDS Clinical Trials Group Genital Secretions
Working Group include Deborah Anderson (Harvard University, Boston,
Mass.), Yvonne Bryson (University of California at Los Angeles),
Lisa Frenkel (University of Washington, Seattle), David Katzenstein
(Stanford University, Stanford, Calif.), Phillip LaRussa (Columbia
University, New York, N.Y.), Jeffrey Lennox (Emory University, Atlanta, Ga.), William A. Meyer III (Quest Diagnostics, Inc., Baltimore, Md.), Cheryl Michels (Data Works Development, Inc., Seattle, Wash.), and Walter Scott (University of Miami, Miami, Fla.).
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Materials and Methods
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Discussion
References
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