Correction of Underquantification of Human Immunodeficiency Virus Type 1 Load with the Second Version of the Roche Cobas AmpliPrep/Cobas TaqMan Assay

Article (PDF Available)inJournal of clinical microbiology 48(4):1337-42 · February 2010with30 Reads
DOI: 10.1128/JCM.01226-09 · Source: PubMed
Abstract
Initial evaluations of the Cobas AmpliPrep/Cobas TaqMan human immunodeficiency virus type 1 (HIV-1) test (CAP/CTM) demonstrated good performance but, afterwards, reports about underquantification were published. We investigated whether the problem was solved with a second version of this assay, the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0). The remaining plasma of 375 consecutive HIV-1 positive samples with a viral load of >or=4,000 copies/ml was collected in three laboratories. The samples were diluted and retested with our routine method Cobas AmpliPrep/Cobas Amplicor HIV-1 monitor test v1.5 in ultrasensitive mode (CAP/CA PHS), as well as with the CAP/CTM and CAP/CTM v2.0 tests. An absolute difference between the results of two methods of >or=0.71 log(10) copies/ml was defined as moderately discrepant, and an absolute difference of >or=0.93 log(10) copies/ml was defined as severely discrepant. In addition, criteria for considering the new methods equivalent to the routine method were formulated. (i) For CAP/CTM compared to CAP/CA PHS, 36 (9.5%) and 20 (5.3%) samples were, respectively, considered moderately and severely underquantified by CAP/CTM. The mean difference between CAP/CTM and CAP/CA PHS was -0.32 log(10) copies/ml. Eight of nineteen of the severely underquantified samples were from patients infected with HIV-1 subtype B strain. (ii) For CAP/CTM v2.0 compared to CAP/CA PHS, no sample was moderately or severely underquantified by CAP/CTM v2.0. A mean difference of 0.08 log(10) copies/ml was found with CAP/CTM v2.0 compared to CAP/CA PHS. The underquantification problem of the CAP/CTM kit was clearly demonstrated. The criteria for the equivalence of CAP/CTM v2.0 to the routine test CAP/CA PHS were fulfilled.

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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2010, p. 1337–1342 Vol. 48, No. 4
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Correction of Underquantification of Human Immunodeficiency Virus
Type 1 Load with the Second Version of the Roche Cobas
AmpliPrep/Cobas TaqMan Assay
A. De Bel,
1
* D. Marissens,
2
L. Debaisieux,
3
C. Liesnard,
3
S. Van den Wijngaert,
2
S. Lauwers,
1
and D. Pie´rard
1
AIDS Reference Laboratory of the Vrije Universiteit Brussel, Subunit Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels,
Belgium
1
; AIDS Reference Laboratory of the Vrije Universiteit Brussel, Subunit Universitair Medisch Centrum Sint Pieter,
Hoogstraat 322, 1000 Brussels, Belgium
2
; and AIDS Reference Laboratory of the Universite´ Libre de Bruxelles,
Erasme University Hospital, Route de Lennik 808, 1070 Brussels, Belgium
3
Received 23 June 2009/Returned for modification 18 September 2009/Accepted 4 February 2010
Initial evaluations of the Cobas AmpliPrep/Cobas TaqMan human immunodeficiency virus type 1 (HIV-1)
test (CAP/CTM) demonstrated good performance but, afterwards, reports about underquantification were
published. We investigated whether the problem was solved with a second version of this assay, the Cobas
AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0). The remaining plasma of 375 consecutive
HIV-1 positive samples with a viral load of >4,000 copies/ml was collected in three laboratories. The samples
were diluted and retested with our routine method Cobas AmpliPrep/Cobas Amplicor HIV-1 monitor test v1.5
in ultrasensitive mode (CAP/CA PHS), as well as with the CAP/CTM and CAP/CTM v2.0 tests. An absolute
difference between the results of two methods of >0.71 log
10
copies/ml was defined as moderately discrepant,
and an absolute difference of >0.93 log
10
copies/ml was defined as severely discrepant. In addition, criteria for
considering the new methods equivalent to the routine method were formulated. (i) For CAP/CTM compared
to CAP/CA PHS, 36 (9.5%) and 20 (5.3%) samples were, respectively, considered moderately and severely
underquantified by CAP/CTM. The mean difference between CAP/CTM and CAP/CA PHS was 0.32 log
10
copies/ml. Eight of nineteen of the severely underquantified samples were from patients infected with HIV-1
subtype B strain. (ii) For CAP/CTM v2.0 compared to CAP/CA PHS, no sample was moderately or severely
underquantified by CAP/CTM v2.0. A mean difference of 0.08 log
10
copies/ml was found with CAP/CTM v2.0
compared to CAP/CA PHS. The underquantification problem of the CAP/CTM kit was clearly demonstrated.
The criteria for the equivalence of CAP/CTM v2.0 to the routine test CAP/CA PHS were fulfilled.
Since the mid-1990, the human immunodeficiency virus type
1 (HIV-1) viral load (VL) assay is a major tool in the follow-up
of HIV-1-infected individuals to predict progression of HIV
disease and to monitor antiviral treatment response (5, 7, 14).
Several patented methodologies using a variety of techniques,
from reverse transcriptase PCR to the branched DNA assay,
are commercially available for quantitative HIV-1 RNA testing
in diagnostic laboratories.
VL assays were developed in industrialized countries, where
HIV-1 subtype B predominates. In contrast, subtype B is a
minor variant in developing countries and dissemination of
non-B subtypes has also started in Western countries, espe-
cially in Belgium, where many viruses of African origin are
circulating (17). Among HIV-1-infected patients attending
Universitair Ziekenhuis Brussel (UZB), as many as 59% of the
strains belong to non-B subtypes (6). This high genetic diver-
sity of HIV-1 is a challenge for the quantification of plasma
HIV-1 RNA. Indeed, in the past, several studies have reported
the failure of commercial assays for viral load monitoring in
patients infected with non-B subtypes (1, 2, 4, 19). This finding
led Roche Diagnostics, Ltd. (Rotkreuz, Switzerland), the man-
ufacturer of the widely used Cobas Amplicor HIV-1 monitor
test version 1.0 assay, to modify this assay in 1997. By the
addition of new primers, the assay could cover a broader range
of viral diversity (Cobas Amplicor HIV-1 monitor test version
1.5 PHS/PHM).
In the early years of 2000, automation in sample extraction
(for example, the Cobas AmpliPrep of Roche Diagnostics and
M1000 of Abbott Diagnostics, Abbott Park, IL) facilitated
high-volume testing and made viral load testing more robust.
With the ongoing introduction of new technologies, classical
endpoint amplification techniques became more and more re-
placed by kinetic real-time fluorescence based tests that com-
bine amplification and detection in one step. A TaqMan-based
real-time technique (10) is simple, rapid, sensitive, specific, and
reproducible and makes further automation possible. More-
over, the risk of contamination is lower due to the closed tube
configuration. The Roche Cobas AmpliPrep/Cobas TaqMan
(CAP/CTM) quantitative HIV-1 assay requires little manual
intervention between the initial addition of the sample to the
assay tube and the generation of the quantitative result. Initial
evaluations of this assay were good (11, 15, 16, 18), but with a
trend to lower viral load values on average than those obtained
with the Cobas Amplicor HIV-1 monitor version 1.5 PHS/
PHM assay (9). Afterward, reports about serious underquan-
* Corresponding author. Mailing address: AIDS Reference Labora-
tory of the Vrije Universiteit Brussel, Subunit UZ Brussel, Laarbeek-
laan 101, 1090 Brussels, Belgium. Phone: 32 2 477 50 00. Fax: 32 2 477
50 15. E-mail: Annelies.DeBel@uzbrussel.be.
Published ahead of print on 17 February 2010.
† The authors have paid a fee to allow immediate free access to
this article.
1337
tification became available (3, 9, 20). The incidence of under-
quantification was low and seemed to be prominent only in
selected populations.
To overcome the issue of underquantification, the CAP/
CTM assay was modified to Cobas AmpliPrep/Cobas TaqMan
HIV-1 test, version 2.0 (CAP/CTM v2.0). A dual-target strat-
egy was chosen: besides gag primers and a FAM-labeled gag
probe, additional ltr primers and a FAM-labeled ltr probe were
included in the assay. The two targets, gag and ltr, are amplified
with the same efficiency. The worst-case scenario is the com-
plete failure of one of the PCR amplicon targets to be ampli-
fied. This would result in the loss of one-half of the fluorescent
signal. The PCR of that specimen would then need one addi-
tional thermal cycle to achieve the threshold cycle (C
T
) for
quantification. Hence, with a C
T
1, the specimen HIV-1
RNA titer would be quantified by half, equivalent to 0.3-log
difference, from the “true” value. Guidelines state that a dif-
ference of 0.5 log copies/ml (cp/ml) is clinically not signifi-
cant (5), and this variation has no clinical consequence.
The aim of the present study was to evaluate if the under-
quantification issue of the CAP/CTM HIV-1 test is solved with
the introduction of the CAP/CTM v2.0 test. A comparison was
performed with the routine Cobas Amplicor HIV-1 monitor
version 1.5, ultrasensitive mode (CAP/CA PHS), the first ver-
sion of CAP/CTM and CAP/CTM v2.0.
(Part of this research was presented at the 7th European
HIV Drug Resistance Workshop, Stockholm, Sweden, 25 to 27
March 2009, poster 86.)
MATERIALS AND METHODS
Samples. From May to September 2008, 375 consecutive EDTA anticoagu-
lated plasma samples (collected in Sarstedt EDTA “lavender” tubes) stored at
below 70°C after acquisition with HIV-1 viral load 4,000 cp/ml in the
CAP/CA PHS (or with manual RNA extraction at Universite´ Libre de Bruxelles
[ULB]) routine test were selected. Apart from this condition and the availability
of a sufficient sample volume, no selection for the inclusion of samples was done.
At UZB, 5 of 83 (6.0%) samples were not included due to insufficient sample
volume. Only one sample per patient was included.
After routine testing of the original specimen the remaining EDTA anticoag-
ulated plasma was diluted 1:5 or 1:10 in HIV-1 RNA negative EDTA plasma
pool (Roche Diagnostics GmbH, Germany) and divided into aliquots. Aliquots
were stored frozen below 70°C until tested. All aliquots were subjected to equal
numbers of freeze-thaw cycles.
Samples were selected and divided into aliquots at 3 laboratories in Brussels:
the AIDS Reference Laboratory of the VUB, subunit UZ Brussel (UZB; n 78)
and subunit University Medical Center Saint-Pierre (STP, n 149) and the
AIDS Reference laboratory of the ULB, Erasme University Hospital (EHB, n
148).
Viral load assays. All samples were analyzed in the AIDS reference laboratory
of the VUB. The subunit UZ Brussel analyzed the samples collected at EHB and
UZB, while the subunit STP tested their own samples.
Each specimen was tested in singlet in the three study tests. The assays were
performed in accordance to the package inserts of the manufacturer. For quan-
tification of the target nucleic acid and control of PCR inhibition an internal
quantification standard was used. The results were expressed in copy number of
HIV-1 RNA copies per milliliter. The prevention of back-contamination was
ensured by the use of uracil-N-glycosylase, an enzyme that inactivates dUTP-
containing amplicons.
(i) Cobas AmpliPrep/Cobas Amplicor HIV-1 monitor test, version 1.5, ultra-
sensitive mode (CAP/CA PHS; Roche Diagnostics, Ltd., Rotkreuz, Switzerland).
After probe-specific extraction with the ultrasensitive protocol on the Cobas
AmpliPrep (CAP), amplification was done with the Cobas Amplicor HIV-1
Monitor Test, v1.5, and endpoint detection was performed using the Cobas
Amplicor (CA) instrument. The primers target the highly conserved gag region.
If the result was described above the upper quantification limit (100,000 cp/ml),
samples were diluted 1:10 or 1:100 with HIV-1 RNA negative human EDTA
plasma and retested to obtain a measurable concentration of HIV-1 RNA.
Several lots of reagents were used to perform the assays.
(ii) Cobas AmpliPrep/Cobas TaqMan HIV-1 test (CAP/CTM; Roche Diagnos-
tics Ltd.). After generic extraction with the CAP, amplification was performed
using the Cobas TaqMan HIV-1 Test on the Cobas TaqMan 48 (CTM) instru-
ment. A dually labeled hybridization probe targeting the gag region was used in
the assay. Multiple kit numbers were used to perform the assays.
(iii) Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0 (CAP/CTM
v2.0; Roche Diagnostics Ltd.). This assay simultaneously targets the gag and the
ltr region with two dually labeled hybridization probes. For the present study, a
prerelease version, CAP/CTM version 1.5 provided by Roche Diagnostics was
used. Afterward, based on extended validation, a software modification was
performed to extend the lower quantification limit from 40 to 20 cp/ml. There-
fore, this test version was later renamed as version 2.0, but the reagents of both
assay versions are the same. This change did not influence our results since no
samples with low viral load were included in this evaluation. CAP/CTM v2.0 is
not yet available in the United States.
After generic extraction with the CAP, amplification was performed using the
Cobas TaqMan HIV-1 Test, v2.0 on the CTM instrument. All assays were
performed using one lot of reagents.
Controls. Each run consisted of 20 samples and one high positive, one low
positive and one negative control included in the kit. In addition, in order to have
an independent control, one in-house produced internal run control was ana-
lyzed in each run.
Criteria for equivalency of two VL methods and discrepancy definitions. The
minimal change in viral load considered as clinically significant is a 0.5 log
10
cp/ml (5). Thus, the maximum total error (TE
MAX
), defined as systematic error
(SE) 1.96 the random error (RE), must be lower than 0.5 log
10
cp/ml.
Because in Belgium HIV patients are monitored at one AIDS reference center
and we know from our validation file that the bias is very small, we can assume
that the SE is negligible. Thus, the maximum allowable RE
MAX
is 0.26 log
10
cp/ml. Because we allow a TE of 0.5 log
10
cp/ml for both methods in a com
-
parison the total TE
MAX
is 1.96
(0.26
2
0.26
2
) or 0.71 log
10
cp/ml. Thus, the
absolute difference between CAP/CA PHS and CAP/CTM or CAP/CTM v2.0
should be 0.71 log
10
cp/ml for at least 95% of all samples. Samples that deviate
by more than 0.71 log
10
cp/ml are referred to as moderately discrepant.
According to the analogous principles described above we can state that for at
least 99% of all samples the absolute difference between CAP/CA PHS and
CAP/CTM or CAP/CTM v2.0 should be less than 2.58
(0.26
2
0.26
2
) or 0.93
log
10
cp/ml. Samples that deviate by more than 0.93 log
10
cp/ml are referred to
as severely discrepant.
Graphs and statistics. Absolute bias plots were used to represent the degree
of agreement between the assays. The x axis of the absolute bias plot shows the
mean of the results, and the y axis represents the absolute difference between the
values obtained by the two platforms. The dotted lines represent the above-
defined criteria for considering the new assay equivalent to the routine assay.
To obtain a normal distribution of the values, results were transferred to log
10
values. Normality was checked by using a modified Shapiro-Wilk test. Mean
values were compared to the t test for paired data. Epidemiological data were
compared to the chi-square test. Statistics were performed with Analyze-It for
Microsoft Excel (version 2.12; Analyze-It Software, Ltd., United Kingdom).
Sequencing and subtyping. Samples with severe discrepancies (n 20) were
sequenced by Roche Molecular Diagnostics (Pleasanton, CA) with unpublished
primers to identify possible mismatches in all primer and probe regions.
Nucleic acids were extracted from 200 l of the original plasma (n 12) or,
when the original plasma was no longer available, from 200 l of the 1:5-diluted
(n 8) plasma using sample preparation reagents from the Cobas HIV-1
Monitor test. A 600-l portion of lysis reagent was mixed with a 200-l sample,
followed by incubation for 10 min at room temperature. Nucleic acids were
precipitated by the addition of 800 l of isopropanol and then pelleted by
centrifugation for 15 min in a microcentrifuge. The pellets were washed with 1 ml
of 70% ethanol (EtOH) and centrifuged for 5 min. The alcohol was removed, the
tubes were pulse spun for 30 s, and the residual EtOH was removed. The pellets
were resuspended in 100 l of sample diluent from the Cobas HIV-1 Monitor
test and stored at 80°C until used.
HIV-1 sequences flanking the CAP/CTM HIV-1 version 2 target regions were
amplified by reverse transcription-PCR. Murine leukemia virus reverse tran-
scriptase (Applied Biosystems, Foster City, CA) was used for reverse transcrip-
tion, and PCR was carried out with AmpliTaq DNA polymerase (Applied Bio-
systems). Heminested amplifications were then performed to increase the yield
of amplicon. Amplification products were analyzed by agarose gel electrophore-
sis. The amplicons from reactions with one predominant product band were
purified for sequencing by using either QiaQuick PCR purification (Qiagen,
1338 DE BEL ET AL. J. CLIN.MICROBIOL.
North Rhine-Westphalia, Germany) or ExoSAP-IT kit reagents (USB, Cleve-
land, OH). If multiple major amplicon populations were present, the products of
appropriate size, separated by agarose gel electrophoresis, were excised from the
gel and purified using reagents from a QiaQuick gel extraction kit (Qiagen).
Sequencing was either performed on the ABI 3730xl analyzer using BigDye
chemistry and Collection Software v3.0 (Applied Biosystems) or by Sequetech
Corp. (Mountain View, CA).
To determine the subtype of the virus in each sample, the gag sequence from
each sample was aligned to sequences from HIV-1 isolates of known genotypes
from the GenBank sequence database (http://www.ncbi.nlm.nih.gov/GenBank
/index.html) using the CLUSTAL V sequence alignment program, which also
generates a phylogenetic tree, via the Lasergene software package (MegAlign
version 5.07; DNASTAR, Inc., Madison, WI).
Demographic information. For each new HIV diagnosis in Belgium the at-
tending physician is asked to fill in a questionnaire and send it to the AIDS
Reference Laboratory that made the diagnosis. All coded data are processed
once a year by the Institute of Public Health to map and characterize the Belgian
HIV epidemic. These data, although not complete, were used to compare the
populations consulting the three different centers and to track the probable
origin of the HIV-1 strains yielding severely discrepant results.
Study ethics. The present study was approved by the biomedical ethical com-
mission of the Universitair Ziekenhuis Brussel and the Medical Faculty of the
Vrije Universiteit Brussel (reference number BUN B14320084122).
RESULTS
Because the initial result was outside the dynamic range of
the CAP/CA PHS assay, 22 samples had to be diluted 1:10 for
the CAP/CA PHS analysis and 1 sample was diluted 1:100 for
additional analysis. No dilutions had to be made for CAP/CTM
and CAP/CTM v2.0 due to the broader dynamic range of these
assays.
One sample (EHB_141) demonstrated an extremely high
quantitative result with both CAP/CTM tests (1.29 and
1.84 log
10
cp/ml) compared to CAP/CA PHS. This result was
considered as an outlier and was not taken into account in our
calculations. The sequence of this sample contains six mis-
matches to the upstream CA PHS primer. The subtype of this
sample was determined as CRF18_cpx.
For one sample (EHB_084) the result for CAP/CTM was
40 cp/ml, although HIV-1 RNA was detected with CAP/CA
PHS (3.70 log
10
cp/ml) and CAP/CTM v2.0 (3.75 log
10
cp/ml).
For the calculations we made an approximation and used 40
cp/ml, the best-case scenario for EHB_084.
CAP/CTM compared to CAP/CA PHS. The results are rep-
resented in an absolute bias plot (Fig. 1).
The overall mean difference between CAP/CTM and
CAP/CA PHS was 0.32 log
10
cp/ml (P 0.05). The median
difference was 0.29 log
10
cp/ml with a range between 2.64
and 0.78 log
10
cp/ml.
A total of 36 samples (9.6%; EHB [n 16], STP [n 15],
and UZB [n 5]) were moderately and 20 samples (5.3%;
EHB [n 10], STP [n 7], and UZB [n 3]) were severely
underquantified with CAP/CTM compared to CAP/CA PHS.
The observed differences between the different sites are not
statistically significant (P 0.55 and 0.59, respectively).
The sequence results and the probable origin of the severely
underquantified strains are represented in Table 1. For one
sample (EHB_039) the amplification of the gag region repeat-
edly failed. The other 19 samples were from patients infected
with 9 different subtypes. Eight of these nineteen (42.1%)
patients were infected with a subtype B strain. In 18 of the
severely underquantified samples, there are mismatches to
CAP/CTM gag primers or probe that may affect assay perfor-
mance. No mismatches were found in one sample (UZB_001),
and thus the observed difference of 1.06 log
10
cp/ml is prob
-
ably due to an anomalous error.
The amplification of the ltr region that was done for further
sequence comparisons succeeded in 16 of these 20 samples.
There were no mismatches found to the CAP/CTM ltr region
that would be likely to affect CAP/CTM v2.0 assay perfor-
mance. Two (0.5%; EHB [n 1], STP [n 1], and UZB [n
0]) samples were moderately higher quantified with CAP/
CTM. These two samples were also higher quantified with
CAP/CTM v2.0. As for sample EHB_141, the sample defined
as an outlier, two (EHB_090) or three (SPB_083) mismatches
to the upstream CA PHS primer were detected.
CAP/CTM v2.0 compared to CAP/CA PHS. The results are
represented in an absolute bias plot (Fig. 2). The titer values
FIG. 1. Modified Bland and Altman plot CAP/CTM versus CAP/CA PHS v1.5. The dotted lines represent the defined acceptance criteria.
V
OL. 48, 2010 CAP/CTM AND CAP/CTM v2.0 ASSAYS FOR HIV-1 VL 1339
obtained with CAP/CTM v2.0 were on average 0.08 log
10
cp/ml higher compared to CAP/CA PHS (P 0.05). The
median difference was 0.05 log
10
cp/ml, with a range from
0.68 to 1.02 log
10
cp/ml. No sample was moderately or se
-
verely underquantified. Seven (1.9%; EHB [n 3], STP [n
3], and UZB [n 1]) samples were moderately and three
(0.8%; EHB [n 1], STP [n 1], and UZB [n 1]) were
severely higher quantified by CAP/CTM v2.0. Five out of seven
moderately higher quantified samples were sequenced. Three
of them had five mismatches to the CA PHS upstream primer.
For two of them no mismatches were found.
Demographic comparison of the patient population attend-
ing the centers. Three parameters (nationality, risk category,
and probable country of infection) were compared by using a
chi-square test. A statistically significant difference (P 0.05)
was observed for all three parameters.
The patient population consulting at UZB contains a higher
fraction of people with the Belgian nationality (44.9% versus
24.2% at STP and 25.2% at EHB). The associated risk cate-
gory is more often men having sex with men (MSM; 44.9%
versus 22.8% at STP and 20.9% at EHB), and the probable
country of infection is more often Belgium (39.4% versus
16.8% at STP and 10.8% at EHB).
The patients seeking medical advice at STP are more often
European (not Belgian), North African, American, and Asian
(11.4% versus 5.1% at UZB and 4.0% at EHB). Moreover, the
risk categories mother-to-child transmission (MTCT) and in-
travenous drug use (IVD) are more prevalent at the STP
patient population (7.4% versus 0.0% at UZB and 2.0% at
EHB).
DISCUSSION
Both American (5) and European (7) guidelines for the
treatment of HIV-1-infected adults make use of two markers—
the viral load and the CD4
T-cell count—to assess the level of
HIV-1 viremia and the immune function of infected patients.
These tests are used as predictors for deciding when to begin
antiviral therapy and to assess virologic and immunologic ef-
ficacy of treatment. Therefore, accurate measurement of
HIV-1 viral load is essential to provide clinicians with valuable
information to determine treatment decisions. Although the
new quantitative HIV-1 assays are designed to cope with in-
creasing molecular diversity of the virus, there were several
reports about serious underquantification issues with the first
version of the CAP/CTM test (3, 9, 20). To overcome this
problem, additional primers and a probe, located in the highly
TABLE 1. Subtype and number of mismatches to gag primers and
probe of the severely underquantified samples
(n 20) in CAP/CTM
Sample Origin Subtype
gag region (no. of mismatches)
Upstream
primer
Downstream
primer
Probe
EHB_003 No data F1 or CRF12_BF 2 5
EHB_025 Sub-Saharan
Africa
D3
EHB_027 No data B 4
EHB_028 Belgium B 1
EHB_039 Sub-Saharan
Africa
AF
a
AF AF AF
EHB_057 Belgium B 1 3
EHB_084 Belgium B 1
EHB_127 Sub-Saharan
Africa
CRF01_AE 1
EHB_131 Sub-Saharan
Africa
K43
EHB_149 No data B 3
SPB_027 Netherlands CRF02_AG 1 1
SPB_042 Sub-Saharan
Africa
H3
SPB_046 Belgium B 1
SPB_072 Sub-Saharan
Africa
A1 1 3
SPB_120 Sub-Saharan
Africa
G3
SPB_122 Sub-Saharan
Africa
G2
SPB_133 Belgium B 2
UZB_001 Sub-Saharan
Africa
D
UZB_026 Sub-Saharan
Africa
F1 or CRF12_BF 1
UZB_039 Belgium B 4
a
AF, amplification failed.
FIG. 2. Modified Bland and Altman plot CAP/CTM v2.0 versus CAP/CA PHS v1.5. The dotted lines represent the defined acceptance cri-
teria.
1340 DE BEL ET AL. J. CLIN.MICROBIOL.
conserved ltr region of HIV-1, were included in the second
version of the kit in addition to the gag primers and probe. We
report here the results of a three-site multicenter evaluation
study of the CAP/CTM and CAP/CTM v2.0 tests in compari-
son to the routine CAP/CA PHS test.
In Belgium, 58% of newly diagnosed HIV patients were of
foreign origin in 2007 (17). Among HIV-1-infected patients
attending UZB, subtype B virus is only present in 41% of the
patients (6). Therefore, the sample collection used was ex-
pected to contain a wide variety of subtypes, including a lot of
non-B subtypes, and consequently could represent a great chal-
lenge for the new generation of viral load tests.
The patient population attending the three centers is signif-
icantly different. The most prominent differences were that the
patient population consulting at UZB contains a higher frac-
tion of MSM with the Belgian nationality and the probable
country of infection is more often Belgium. The patients seek-
ing medical care at STP are more often from European (non-
Belgian), North African, American, and Asian nationalities.
Moreover, the risk categories MTCT and IVD are more prev-
alent at the STP patient population.
The results of the comparison of CAP/CTM and CAP/CA
PHS are represented in an absolute bias plot (Fig. 1). A total
of 36 of 375 (9.6%) samples were moderately and 20 of 375
(5.3%) samples were severely underquantified. Although not
statistically significant (P 0.59), the prevalence of severe
underquantification was highest at EHB: 6.7% of samples
compared to 4.6% at STP and 3.8% at UZB. No particular
subtype is affected by the underquantification problem. The 19
subtyped, severely discrepant samples were from patients in-
fected with nine different subtypes. Eight of these nineteen
(42.1%) patients were infected with a subtype B strain. Thus,
in contrast to earlier-generation HIV-1 VL assay problems (1,
2, 4, 19), even subtype B strains could be affected by the
underquantification issue of the first version of the CAP/CTM
test, indicating that the mismatches were not associated with
the HIV-1 subtype.
As illustrated in Table 1, both the primer and the probe
binding region polymorphisms were identified as the root
cause in the rare cases of significant underquantification. Due
to patented primers and probes, Roche did not wish to disclose
the exact location of the mismatches. Therefore, we cannot
support or refute the theory of Korn et al. (12) that mutations
at nucleotide position 1488 of the HXB2 reference sequence,
the 3 position of the suggested downstream primer, are an
important cause of the underestimation of HIV-1 RNA levels.
Nevertheless, there should be other critical mutations because
8 of the 19 sequenced underquantified samples had no mis-
matches to the downstream primer.
We found that 2 of 375 (0.5%) samples were moderately
higher quantified by CAP/CTM. These two samples are also
moderately higher quantified (0.79 and 0.91 log
10
cp/ml,
respectively) with CAP/CTM v2.0. Although not well docu-
mented since we only had access to the number of mismatches
and not to the sequences as such, this finding is probably due
to a better primer-probe match than with the CAP/CA PHS
assay, the method used as the “reference” or comparison
method. This is also the case with sample EHB_141, which we
considered an outlier.
The overall mean difference between CAP/CTM and
CAP/CA PHS was 0.32 log
10
cp/ml (P 0.05). This differ
-
ence is statistically significant. Although this difference is 0.5
log
10
cp/ml, which is generally accepted as clinically relevant
(5), it clearly illustrates the underquantification issue of the
first version of CAP/CTM. Moreover, the high number of
moderately and severely underquantified samples is clinically
problematic. The criteria used above to consider the new
method equivalent to the routine method are not fulfilled for
CAP/CTM.
The results of the comparison of CAP/CTM v2.0 and
CAP/CA PHS are represented in an absolute bias plot (Fig. 2).
No sample was moderately or severely underquantified. Eight
(2.1%) samples were moderately and three (0.8%) of 375 sam-
ples were severely higher quantified with CAP/CTM v2.0. In
all, four samples (three EHB_141) had several mismatches
to the upstream CA PHS primer and were probably under-
quantified with the older CAP/CA method. Thus, it appears
that the high genetic diversity of HIV-1 also affects the per-
formance of the “reference” or comparison method.
A mean difference of 0.08 log
10
cp/ml was found with the
CAP/CTM v2.0 compared to CAP/CA PHS (P 0.05). This
difference is statistically significant but not clinically relevant
(0.5 log
10
cp/ml). The above-described criteria to consider
the new method equivalent to the routine method are fulfilled
for CAP/CTM v2.0.
In addition, one sample (EHB_141) quantified extremely
high with both Cobas TaqMan tests (1.29 and 1.84 log
10
cp/ml; CAP/CTM and CAP/CTM v2.0, respectively) compared
to the routine CAP/CA PHS test and was considered to be an
outlier. The patient’s CD4 count and clinical presentation cor-
related better with the higher VL (this patient stopped taking
all antiretroviral therapy at sampling time). The sequence of
the HIV-1 strain from this sample revealed six mismatches to
the upstream CA PHS primer. Therefore, we consider that it
was underquantified by the CAP/CA PHS test.
The present study has three limitations. (i) Lot-to-lot vari-
ability for the CAP/CTM v2.0 kit was not evaluated during our
study. Only one lot of reagents was used during the evaluation.
(ii) A bias was possibly generated by selecting only samples
withaVLof4,000 copies/ml, which were diluted in order to
obtain enough sample volume to run the three tests in parallel.
Indeed, most of the included samples were from untreated
patients, harboring viral strains without mutation pressure
from antiretroviral drugs. However, gag and ltr are not current
drug targets and, although gag cleavage site mutations have
been described to be involved in protease inhibitor resistance
(21), all known gag mutations associated with drug resistance
lie outside the gag PCR amplicon target region used by the
CAP/CTM assays (S. Rose, Roche Molecular Diagnostics, per-
sonal communication). Hence, mutations associated with drug
resistance should not affect the test performance. Moreover,
dilution with HIV-1 RNA negative EDTA plasma might have
influenced the efficiency of the extraction and amplification.
Nevertheless, even with a possibly biased sample pool, we were
able to clearly demonstrate the underquantification problem
with CAP/CTM. (iii) The present study did not evaluate the
performance of the tests in the low quantification range. In
order to find significant differences in quantification, speci-
mens with higher nominal viral loads were used. However, as
seen with the first version of this kit (16, 18), the second version
VOL. 48, 2010 CAP/CTM AND CAP/CTM v2.0 ASSAYS FOR HIV-1 VL 1341
will probably also show an increased sensitivity at low viral
loads in comparison with the CAP/CA PHS assay, as this fact
is linked to the real-time technology. The important practical
impact of this issue was recently discussed (8, 13).
In conclusion, the criteria needed to consider the new
method equivalent to the routine method are fulfilled only for
CAP/CTM v2.0. We clearly demonstrated the underquantifi-
cation issue with the first version of the CAP/CTM test, not
only in 13 samples of patients infected with a non-B subtype
but also in 7 samples from patients infected with subtype B.
Although the samples tested are all of Belgian origin (or at
least collected in Belgium), the CAP/CTM test is not adequate
for HIV-1 viral load testing on a general, worldwide basis.
With the CAP/CTM v2.0 test no underquantified sample was
identified compared to the routine test CAP/CA PHS. Thus,
only the second version of this kit can be used for routine
HIV-1 viral load testing in a routine clinical laboratory.
In spite of the good correlation between CAP/CA PHS and
CAP/CTM v2.0, clinical biologists and clinicians should remain
aware of the high degree of genetic variability of HIV-1 and
the difficulties that this entails in the design of appropriate
primer and probe sets for a real-time PCR that should be able
to quantify all known HIV-1 variants. When viral load status
does not develop as expected from clinical picture and/or
CD4
T-cell counts, other plasma HIV-1 RNA assays should
be used in order to detect possible variation of the virus,
whatever its subtype or origin.
ACKNOWLEDGMENTS
We thank Roche Diagnostics, Ltd., Rotkreuz, Switzerland for sup-
plying the reagents used in this study, for sequencing the severely
discrepant samples and for lending us the Cobas TaqMan 48 analyzer.
We gratefully acknowledge A. Wyns, C. Vanneste, M.-H. Jurion, K.
Miller, and N. Gijbels for their excellent technical assistance. We thank
A. Sasse from the Belgian Institute of Public Health for his support.
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1342 DE BEL ET AL. J. CLIN.MICROBIOL.
    • "SCA is more sensitive than any of the clinical detection methods that are currently available. Commercial assays including the Roche Ampliprep [30] and Abbot real time HIV-1 assays can also be used to measure HIV-1 RNA in the plasma. These assays are much less sensitive than the SCA with lower limits of 20 or 40 copies per milliliter, respectively. "
    [Show abstract] [Hide abstract] ABSTRACT: HIV is a devastating worldwide epidemic that has had substantial social and economic impacts throughout the globe. Due to the presence of a small pool of latently infected cells that persists during antiretroviral therapy (ART), HIV is not curable. Because of the high cost of ART and the lack of reliable accessibility across the globe, life-long ART is unfortunately not a feasible solution for the epidemic. Therefore, new strategies need to be developed and implemented to address HIV-1 infection. Several approaches toward this end are currently under investigation (Ebina et al. in Sci Rep 3:2510, 2013; Archin et al. in Nature 487:482-5, 2012; Elliott et al. in PLoS Pathog 10:e1004473, 2014; Rasmussen et al. in Lancet HIV 1:e13-e21, 2014; Tebas et al. in N Engl J Med 370:901-10, 2014; Archin et al. in Nat Rev Microbiol 12:750-64, 2014; Barton et al. in PLoS One 9:e102684, 2014; Sogaard et al. in PLoS Pathog 11:e1005142, 2015). Initial studies have proven promising, but have highlighted the need for sensitive and accurate assays to detect changes in very low concentrations of virus to allow confident interpretation of the success of curative approaches. This review will focus on assays that are currently available and the advantages and limitations of each.
    Full-text · Article · Feb 2016
    • "As viruses were cultured to high-titer/high-volume, they were fully characterized including final viral load, p24, TCID, sterility testing, full length sequencing, and coreceptor analysis. A summary of the current EQAPOL panel is provided inTable 2. Viral load was quantified using the Roche COBAS Taqman Version 2.0 assay, which can quantify a broad range of subtypes, including Group O (De Bel et al., 2010). The average culture supernatant final product concentration was 4.20 × 10 9 cp/mL, and the average p24 was 206.59 ng/mL (Table 3). "
    [Show abstract] [Hide abstract] ABSTRACT: The significant diversity among HIV-1 variants poses serious challenges for vaccine development and for developing sensitive assays for screening, surveillance, diagnosis and clinical management. Recognizing a need to develop a panel of HIV representing the current genetic and geographic diversity NIH/NIAID contracted the External Quality Assurance Program Oversight Laboratory (EQAPOL) to isolate, characterize and establish panels of HIV-1 strains representing global diverse subtypes and circulating recombinant forms (CRFs), and to make them available to the research community. HIV-positive plasma specimens and previously established isolates were collected through a variety of collaborations with a preference for samples from acutely/recently infected persons diagnosed since 2009. Source specimens were cultured to high-titer/high-volume using well-characterized cryopreserved PBMCs from healthy donors. Panel samples were stored as neat culture supernatant or diluted into defibrinated plasma. Characterization for the final expanded virus stocks included viral load, p24 antigen, infectivity (TCID), sterility, coreceptor usage, and near full-length genome sequencing. Viruses are made available to approved, interested laboratories using an online ordering application. The current EQAPOL Viral Diversity panel includes over 101 viral specimens representing 6 subtypes (A, B, C, D, F, and G), 2 sub-subtypes (F1 and F2), 7 CRFs (01, 02, 04, 14, 22, 24, and 47), 19 URFs and 3 group O viruses from 22 countries. The EQAPOL Viral Diversity panel is an invaluable collection of well-characterized reagents that are available to the scientific community, including researchers, epidemiologists, and commercial manufacturers of diagnostics and pharmaceuticals to support HIV research and diagnostic and vaccine development.
    Full-text · Article · Jan 2014
    • "This study also confirmed an unrelated but potentially more serious shortcoming of the TaqMan v1 assay. The TaqMan v1 primers were unable to efficiently amplify their targets in a subset of ,2.4% of samples tested leading to the systematic underestimation of viral loads by up to 2.5 log 10 copies/mL as confirmed by re-testing by Amplicor v1.5, TaqMan v2, and/or the Abbott RealTime assays (see also [9,15,25]). When HIV-1 gag sequences from these samples were sent to Roche for analysis, 78% were identified as having mutations incompatible with the TaqMan v1 primers. "
    [Show abstract] [Hide abstract] ABSTRACT: The lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility. Sudden increases in the number of patients with detectable pVL have been reported following the introduction of the TaqMan version 1 assay. Between October 2009 and April 2010 all routine pVL samples from British Columbia, Canada, with 40-250 copies/mL by TaqMan were re-tested by Amplicor (N = 1198). Subsequent short-term virological and resistance outcomes were followed in patients with unchanged therapy (N = 279; median 3.2 months follow-up). TaqMan and Amplicor values correlated poorly at low pVL values. Low-level pVL by TaqMan was not associated with impending short-term virological failure; only 17% of patients with 40-250 copies/mL by TaqMan had detectable pVL by Amplicor at follow-up. During the follow-up period only 20% of patients had an increase in pVL by TaqMan (median [IQR]: 80 [36-283] copies/mL). In addition, in ~2.4% of samples pVL was dramatically underestimated by TaqMan due to poor binding of the proprietary TaqMan primers. The replacement of Amplicor with the TaqMan assay has altered the previously accepted definition of HIV treatment failure without any evidence to support the clinical relevance of the new definition. Given the systematic differences in measurement in the low pVL range the British Columbia HIV treatment guidelines now use a threshold of >250 copies/mL by TaqMan to define treatment failure.
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