1
Comparative Evaluation of the ExaVirTM
Load Version 3 Reverse Transcriptase 1
Assay for the Measurement of HIV-1 Plasma Viral Load 2
Wendy Labbett1, Ana Garcia-Diaz1, Zoe Fox2, Gillian S. Clewley1, Thomas Fernandez3, 3
Margaret Johnson3, and Anna Maria Geretti1,3*. 1Department of Virology, 2Department of 4
Infection and Population Health, and 3Department of HIV Medicine, Royal Free 5
Hampstead NHS Trust and University College London Medical School, London, United 6
Kingdom 7
8
9
*Corresponding author 10
Mailing address: Department of Virology, 11
Royal Free Hampstead NHS Trust & UCL Medical School 12
Pond Street, London NW3 2QG, United Kingdom 13
Phone: +44 20 7317 7521 14
Fax: +44 20 7830 2854 15
E-mail: [email protected] 16
17
Running title: ExaVir HIV-1 plasma RNA load assay 18
Key words: HIV, viral load, RT assay, real-time PCR 19
20
The work was presented at the 16th Conference on Retroviruses and Opportunistic 21
Infections, Montreal 8-11 February 2009. 22
Supported by the Royal Free Hampstead NHS Trust Departmental R&D Fund 23
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00715-09 JCM Accepts, published online ahead of print on 5 August 2009
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Abstract 24
Background: In resource-limited settings, virological monitoring of antiretroviral therapy is 25
limited by high cost and lack of infrastructure. The Cavidi ExaVirTM Load assay employs a 26
simple and inexpensive ELISA format to measure HIV reverse transcriptase activity, which 27
correlates with plasma RNA load. The version 3 assay has been described as having 28
improved precision and sensitivity. There are limited data on its performance relative to 29
current real-time assays. 30
Objective: To compare HIV-1 RNA load measurement in plasma by ExaVirTM Load v.3 31
(“ExaVir”), Abbott M2000sp/M2000rt RealTime HIV-1 (“RealTime”) and Roche COBAS 32
Ampliprep/COBAS TaqMan HIV-1 v.1 (“TaqMan"). 33
Methods: Plasma from 119 patients (subtype B 34, non-B 85: A-H, CRF01, CRF02, 34
CRF06, CRF12, CRF14, complex; treatment experienced 48, naive 71) and serial dilutions 35
of the 2nd International Standard (IS) were tested. Assay relationship and agreement were 36
determined by linear regression, correlation analysis and the Bland-Altman method. 37
Results: ExaVir quantified 77/83 (92.8%) samples with viral load >2.3 log10 copies/ml by 38
the molecular assays. Results were linearly associated and strongly correlated with 39
RealTime and TaqMan measurements (R 0.94, 0.92), for both B (R 0.97, 0.95) and non-B 40
(R 0.93, 0.91) subtypes. Mean differences were 0.28 and 0.18 log10 copies/ml in favour of 41
the two molecular assays; 7/119 (5.9%) and 5/119 (4.2%) samples were outside the 95% 42
level of agreement. ExaVir under-quantified the IS by mean 0.2 (range 0.0, 0.5) log10 43
copies/ml. 44
Conclusion: The ExaVirTM Load v.3 assay showed excellent concordance with real-time 45
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molecular assays, offering a suitable option for virological monitoring in settings with 46
limited infrastructure. 47
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Introduction 48
The introduction of combination antiretroviral therapy in resource-limited countries has 49
resulted in significant reductions in morbidity and mortality [8, 13, 22]. It is generally 50
accepted that unavailability of plasma viral load (VL) monitoring should not preclude 51
expanded access to treatment in these settings [19]. However, knowledge accrued through 52
over a decade of experience in high-income settings indicates that virological monitoring is 53
required in treated patients to ensure optimal long-term outcomes [2, 11]. Incomplete VL 54
suppression during therapy leads to the emergence and evolution of drug-resistance, 55
reducing treatment options and resulting in the transmission of resistant mutants [7]. 56
Neither clinical findings nor CD4 cell counts are adequate predictors of viral suppression, 57
and in fact, management by CD4 cell counts alone can lead to unnecessary treatment 58
changes [1]. VL testing is the only reliable marker for the early detection of failure of 59
antiretroviral therapy [17, 20]. 60
61
Molecular VL assays in routine use in high-income countries require expensive instruments 62
and reagents, sophisticated laboratory facilities to minimise the risk of contamination, 63
regular and stable electricity supply, and highly skilled laboratory technicians proficient in 64
molecular biology techniques. These factors limit the implementation of VL testing in 65
resource-limited settings. The Cavidi ExaVirTM Load assay employs a modified ELISA 66
format to measure the viral reverse transcriptase (RT) activity, which in turn correlates with 67
plasma RNA levels [3, 23]. The assay requires simple, routinely available equipment (e.g., 68
incubator, ELISA-plate reader, freezer, mixing table and vortex) and is relatively 69
inexpensive and simple to perform. The price per test is dependent on volumes but can be 70
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as low as $13.66 (Personal communication from Martyn Eales, Cavidi, Sweden). These 71
characteristics make it suitable for use in settings with limited infrastructure. In November 72
2007, the manufacturer signed an agreement with the Clinton Foundation HIV/AIDS 73
Initiative (CHAI) to provide the assay at a discounted price to members of the CHAI 74
Procurement Consortium of over 70 developing countries. The two previous versions of the 75
assay have been evaluated in the literature [3, 5, 12, 14, 15, 23, 25]. Relative to version 2, 76
version 3 is described by the manufacturers as showing enhanced precision, analytical 77
specificity and sensitivity (lower limit of quantification lowered from 400 to 200 78
copies/ml), improved turn-around time (from 72 to 48 hours), reduced hands-on time (from 79
6 to 5 hours) and use of consumables, and increased through-put (from 120 to 180 samples 80
per week per scientist). There is no published evidence on the performance of the version 3 81
assay relative to current real-time molecular methods in use in high-income countries. 82
83
The objective of this study was to evaluate the performance of the ExaVirTM Load v.3 assay 84
(referred to as the ExaVir assay) in comparison with two real-time PCR assays widely used 85
in high-income countries: the Abbott M2000sp/M2000rt RealTime HIV-1 assay (referred to 86
as the RealTime assay) and the Roche COBAS-Ampliprep/COBAS-TaqMan HIV-1 v.1 87
assay (referred to as the TaqMan assay). 88
89
Materials and Methods 90
Patient and samples 91
Blood samples anticoagulated with EDTA were collected from 119 patients attending the 92
Royal Free Hampstead NHS Trust for routine HIV care. The study population was infected 93
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with diverse HIV-1 subtypes and comprised 71 antiretroviral treatment-naive patients and 94
48 patients receiving antiretroviral therapy. Plasma was separated within 6 hours of 95
collection and stored at -80°C in three separate aliquots, prior to parallel testing in the three 96
assays. Serial dilutions (n=10) ranging from 4.4 to 1.6 log10 (25,000 to 40) IU/ml of the 97
World Health Organization (WHO) subtype B 2nd International Standard for HIV-1 RNA 98
(IS) (National Institute for Biological Standards and Control, UK, product code 97/650) 99
were also tested in parallel. Plasma samples from 10 HIV antibody-negative patients were 100
used as negative controls to assess ExaVir specificity. 101
102
ExaVir assay 103
The ExaVirTM Load assay (Cavidi, Sweden) measures the viral RT enzymatic activity in an 104
ELISA format. Following separation of virus particles from 1 ml of plasma using a solid 105
phase extraction manifold, virus is lysed to obtain the RT enzyme, and the lysate is added 106
to an RNA template bound to the solid phase in the presence of primer and RT substrate. In 107
the presence of RT, the enzyme synthesizes a DNA strand, which is detected by α-BrdUm 108
monoclonal antibody conjugated to alkaline phosphatise (AP). The product is quantified by 109
the addition of a colorimetric AP substrate. The RT activity in the sample is determined by 110
the ExaVir Load Analyzer software through a standard curve generated by an eleven point 111
serial dilution of a known amount of recombinant HIV-1 RT. The range of quantification, 112
as reported by the manufacturers, is from approximately 200 (2.3 log10) to 600,000 (5.8 113
log10) copies/ml. The upper limit varies with the reading range of the ELISA plate-reader. 114
RealTime assay and TaqMan assay 115
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The Abbott RealTime HIV-1 assay (Abbott Diagnostics, USA) and the Roche COBAS-116
TaqMan HIV-1 v.1 assay (Roche Molecular Diagnostics, Germany) employ high 117
throughput, automated real-time PCR methodologies targeting conserved regions in HIV-1 118
integrase and gag respectively. The RNA is extracted from 1 ml of plasma and concentrated 119
using magnetic particle technology in the automated Abbott M2000sp instrument and the 120
Roche COBAS Ampliprep instrument. Detection and quantification of the amplified PCR 121
product is accomplished within hours by monitoring the emission intensity of fluorescent 122
reporter dyes released during the amplification process. The reported range of 123
quantification with is 40 to 10,000,000 (1.6, 7.0 log10) copies/ml. 124
125
HIV-1 subtyping 126
HIV-1 subtypes were determined from pol gene sequences using the ViroSeq system 127
(Celera Diagnostics, USA). Briefly, following reverse transcription of plasma RNA, a 128
1.8kb amplicon comprising the whole of protease and codons 1-335 of RT underwent 129
population sequencing in an ABI PRISM 3100 genetic analyzer. The sequences were 130
submitted to the NCBI and Rega HIV-1 subtyping tools and the assignment was confirmed 131
by phylogenetic analysis with Mega 4.0 using references sequences from the Los Alamos 132
database (www.lanl.gov). 133
134
Statistical analysis 135
VL measurements were log10 transformed before analysis and the value of the assay lower 136
limit of quantification (LLQ) was assigned to samples with VL below this level. Pair-wise 137
Pearson's correlation coefficients were used to assess whether VL values determined using 138
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different assays were correlated. Since correlation coefficients do not account for the fact 139
that one assay may provide consistently higher values compared to another assay, pair-wise 140
Bland-Altman plots were used to further assess the level of agreement. These plots compare 141
two measurement techniques by plotting the difference in VL measurements between any 142
two assays against the average of the two assays. These differences were then tested using 143
paired t-tests for each pair-wise comparison. The Pitman’s test, based on calculating the 144
correlation between the difference and the mean, was used to test for a null hypothesis of 145
equal variances given bivariate normality. The t-test was also repeated assuming unequal 146
variances (and unpaired data), with similar results (not shown). 147
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Results 148
Patient samples 149
The 119 samples comprised HIV-1 group M strains representing 34 subtypes B and 85 150
diverse non-B subtypes (Table 1). At the time of sampling, 71 patients were antiretroviral 151
treatment naive and 48 were receiving antiretroviral therapy. 152
153
Comparison of the ExaVir assay with the RealTime assay 154
Overall, 78/119 (65.5%) samples were quantified by both assays with a median VL of 4.4 155
(range 2.4, 5.8) log10 copies/ml by ExaVir and 4.6 (range 2.4, 6.8) log10 copies/ml by 156
RealTime. There were 25/119 (21.0%) samples with undetectable VL by both assays, all 157
from treated patients. A further 15/119 (12.6%) samples with a median VL of 2.3 (range 158
1.6, 3.3) log10 copies/ml by RealTime showed an undetectable VL by ExaVir, including 159
seven samples that were quantified by RealTime at levels above the expected LLQ of 160
ExaVir (2.3 log10, 200 copies/ml). One sample (1/119, 0.8%) (subtype D, on antiretroviral 161
therapy) showed a VL of 3.1 log10 (1259) copies/ml by ExaVir but an undetectable VL by 162
RealTime. The coefficient of correlation R between the assays was 0.94 overall, and 0.97 163
and 0.93 for B and non-B subtypes respectively (Figure 1a). The VL measurements differed 164
on average by 0.28 (95% confidence interval, CI 0.19, 0.37) log10 copies/ml in favour of 165
RealTime (P<0.0001). Pitman's test difference in variance (r) = 0.107 (p=0.248). In the 166
Bland-Altman comparison, the limits of agreement (reference range for difference) were -167
0.72 to 1.27 log10 copies/ml (Figure 2a). Overall 7/119 (5.9%) samples (3 CRF02, 2 168
subtype D, 1 subtype A, 1 subtype B) fell outside the reference range, including six samples 169
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under-quantified by ExaVir and the subtype D sample quantified by ExaVir but not by 170
RealTime (Table 2). 171
Comparison of the ExaVir assay with the TaqMan assay 172
Overall, 77/119 (64.7%) samples were quantified by both assays with a median VL of 4.4 173
(range 2.4, 5.8) log10 copies/ml by ExaVir and 4.5 (range 2.8, 6.8) log10 copies/ml by 174
TaqMan. There were 25/119 (21.0%) samples with undetectable VL by both assays, all 175
from treated patients. A further 15/119 (12.6%) samples with a median VL of 2.3 (range 176
1.8, 3.2) log10 copies/ml by TaqMan showed an undetectable VL by ExaVir, including 177
seven samples that were quantified by TaqMan at levels above the expected LLQ of 178
ExaVir. Two samples (2/119, 1.7%) (subtype D, CRF01) showed a VL of 3.1 and 3.9 log10 179
copies/ml respectively by ExaVir but an undetectable VL by TaqMan. By RealTime, the 180
subtype D sample from a treated patient also showed an undetectable VL, whereas the 181
CRF01 sample showed a VL of 3.2 log10 copies/ml. The coefficient of correlation R 182
between the two assays was 0.92 overall, and 0.95 and 0.91 for B and non-B subtypes 183
respectively (Figure 1b). The VL measurements differed on average by 0.18 (95% CI: 0.08, 184
0.29) log10 copies/ml in favour of TaqMan (P=0.0005). Pitman's test of difference in 185
variance: r = -0.043 (p=0.647). In the Bland-Altman comparison, the limits of agreement 186
(reference range for difference) were -0.94 to 1.31 log10 copies/ml (Figure 2b). Overall, 187
5/119 (4.2%) samples (2 CRF02, 1 subtype D, 1 CRF01, 1 CRF14) fell outside the 188
reference range, including two samples (CRF02) under-quantified by ExaVir, and three 189
samples (subtype D, CRF01, and CRF14) under-quantified by the TaqMan assay (Table 190
2). 191
192
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Comparison of the RealTime assay with the TaqMan assay 193
Overall 91/119 (76.5%) samples were quantified by both assays with a median VL of 4.5 194
(range 1.6, 6.8) log10 copies/ml by RealTime and 4.4 (range 1.8, 6.8) log10 copies/ml by 195
TaqMan. There were 25/119 (21.0%) samples with undetectable VL by both assays, all 196
from treated patients. In addition, one sample (1/120, 0.8%) (subtype A) showed a VL of 197
2.2 log10 copies/ml by TaqMan but an undetectable VL by RealTime. Conversely, two 198
samples (2/119, 1.7%) (subtype C, CRF01) showed a VL of 2.3 and 3.2 log10 copies/ml 199
respectively by RealTime but an undetectable VL by TaqMan. The coefficient of 200
correlation R was 0.96 overall, and 0.97 and 0.96 for B and non-B subtypes respectively 201
(Figure 1c). The VL measurements differed on average by 0.09 (95% CI 0.02, 0.17) log10 202
copies/ml in favour of RealTime (P=0.01). Pitman's test of difference in variance: r = 0.194 203
(p=0.035). In the Bland-Altman comparison, the limits of agreement (reference range for 204
difference) were -0.69 to 0.88 log10 copies/ml (Figure 2c). Overall 6/119 (5.0%) samples 205
fell outside the reference range. comprising five samples (1 subtype B, 1 subtype D, 1 206
CRF01, 1 CRF02, 1 CRF14) under-quantified by TaqMan and 1 sample (subtype D) under-207
quantified by RealTime (Table 2). 208
209
Assay performance with the WHO International HIV-1 RNA Standard 210
ExaVir consistently under-quantified the IS, whereas the TaqMan assay consistently over-211
quantified the subtype B IS (Figure 3). With ExaVir, across seven quantified dilutions 212
ranging from 4.4 to 2.6 log10 IU/ml, the mean difference in VL was 0.3 (range 0.0, 0.5) 213
log10 copies/ml. With the molecular assays, across 10 dilutions ranging from 4.4 to 1.6 log10 214
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IU/ml, the average difference was 0.0 (range 0.1, -0.3) log10 copies/ml with RealTime and -215
0.2 (range -0.1, -0.3) log10 copies/ml with TaqMan. 216
217
Reproducibility and specificity of the ExaVir assay 218
To assess the intra-assay reproducibility of the EvaVir assay, 10 samples were tested in 219
duplicate, of which seven showed a detectable VL. Overall replicate values differed by 220
mean 0.04 (standard error 0.07) log10 copies/ml. Specificity was assessed with 10 HIV-221
negative plasma samples, all of which showed an undetectable VL. 222
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Discussion 223
Molecular VL assays widely used in high-income countries for virological monitoring of 224
antiretroviral therapy are difficult to implement in resource-limited settings due to both 225
financial and practical constraints. The ExaVir assay offers a cheaper and simpler 226
methodology for VL measurement in these settings. In this study, the version 3 assay 227
showed an excellent correlation and a high degree of concordance with two widely used 228
commercial real-time PCR assays, a comparative performance similar to the relationship 229
that the two molecular assays showed with each other. 230
231
Previous studies analysed the performance of the Exavir versions 1 and 2 and found good 232
detection rates for samples with viral load above 10,000 and 400 copies/ml respectively, 233
and a good overall correlation with molecular assays, most commonly the Roche Amplicor 234
HIV-1 Monitor Test v1.5 [3, 5, 12, 14, 15, 23, 25]. An evaluation of the performance of the 235
version 3 assay in relation to version 2 (and the Roche Amplicor HIV-1 Monitor Test v1.5) 236
was presented in abstract form in 2008 [9]. It demonstrated increased sensitivity with 237
version 3 relative to version 2, with a mean difference 0.19 log10 copies/ml. In this study, 238
VL measurements with clinical samples were generally under-quantified by ExaVir version 239
3 relative to the molecular assays. With the IS, we also observed under-quantification by 240
ExaVir, while detecting good performance of RealTime and a small but consistent over-241
quantification by TaqMan. The ExaVir assay quantified 93% of samples with VL >2.3 242
log10 (>200) copies/ml by both molecular assay, and 100% of samples with VL >3.2 (1585) 243
to 3.3 (1995) log10 copies/ml. Thus, performance was overall in agreement with the range 244
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of quantification reported by the manufacturers. These findings indicate that ExaVir can 245
reliably identify significant viremia in treated patients. 246
247
The significance of low-level viremia continues to be debated in high-income countries [16, 248
18]. Interpretation and management are likely to be even more challenging in developing 249
countries where drug options are limited. Previous studies reported stable CD4 cell counts 250
and a low risk of disease progression at VL levels below 4.0 log10 copies/ml [4, 18]. 251
However, it would be of importance to consider additional outcome data, including 252
emergence of drug-resistance and exhaustion of treatment options, in order to establish 253
appropriate VL cut-offs that should trigger a treatment change where resources are limited. 254
Meanwhile, an assay with a lower limit of quantification of around 200 copies/ml would be 255
of immediate practical use. 256
257
A few samples showed significant difference in VL measurements between assays. ExaVir 258
generally under-quantified these discrepant samples relative to the molecular assays, 259
consistent with the reduced sensitivity of the assay. While a problem with false positive 260
results was apparent in a study of the ExaVir version 2 assay [24], there was no evidence of 261
a significant problem with assay specificity in this study. Among 26 samples with an 262
undetectable VL by the two molecular assays, all from treated patients, only one showed a 263
detectable VL by ExaVir, at 3.1 log10 (1259) copies/ml, while HIV negative samples all 264
showed an undetectable VL by ExaVir. One additional sample, from a patient infected with 265
CRF01 was quantified by ExaVir as well as RealTime, but not by TaqMan, suggesting a 266
problem with quantification by the latter. Although RealTime and Taqman showed a high 267
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degree of concordance, four other samples, comprising subtype B, subtype D, CRF02 and 268
CRF14, were significantly under-quantified by TaqMan, whereas one subtype D sample 269
was under-quantified by RealTime. Previous studies suggested an impaired performance of 270
the TaqMan v.1 assay for the quantification of non-B subtypes [10, 21] and the recently 271
launched v.2 assay promises to address this problem. We previously reported good overall 272
performance of the RealTime assay [6]. In this study, underperformance of either real-time 273
PCR assay was more common with non-B subtypes, but not consistent with specific 274
subtypes. Taken together these findings indicate that, despite the significant improvements 275
introduced in recent years, HIV sequence variability continues to challenge molecular VL 276
assays. Testing with a second method is recommended when VL results are not consistent 277
with the patient’s history and in these circumstances, the use of a non-molecular assay like 278
the ExaVir could be also considered. 279
280
In summary, we found an excellent correlation and a high degree of concordance between 281
the ExaVirTM Load v.3 assay and current real-time molecular assays. The increased 282
through-put and reduced turn-around time, hands-on time and use of consumables with v.3 283
relative to v.2 make the assay an attractive option for virological monitoring of treated 284
patients where infrastructure is limited. Some of the previously recognised limitations [25] 285
remain, including the large sample volume required for analysis and the lack of automation. 286
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Acknowledgments 287
We wish to thank Martyn Eales at Cavidi, Sweden, for assistance with setting up the 288
ExaVir assay. 289
290
Financial disclosure 291
W.L. and G.C. have received travel support from Abbott Diagnostics. 292
M.J. has received consultancy and speaker honoraria from Abbott Pharmaceuticals and 293
Roche Pharmaceuticals. 294
A.M.G. has received consultancy and speaker honoraria from Abbott Diagnostics, Abbott 295
Pharmaceuticals, Roche Molecular Diagnostics and Roche Pharmaceuticals. 296
297
References 298
1. Bagchi, S., M.C. Kempf, A.O. Westfall, A. Maherya, J. Willig, and M.S. Saag. 2006. 299
Can routine clinical markers be used longitudinally to monitor antiretroviral therapy 300
success in resource-limited settings? Clin. Infect. Dis. 44:135-138. 301
2. Bracciale, L., S. Di Giambenedetto, M. Colafigli, G. La Torre, M. Prosperi, R. 302
Santangelo,S. Marchetti, R. Cauda, G. Fadda, and A. De Luca. 2009. Virological 303
suppression reduces clinical progression in patients with multiclass-resistant HIV type 304
1. AIDS Res Hum Retroviruses. 25:261-267. 305
3. Braun, J., J.C. Plantier, M.F. Hellot, E. Tuaillon, M. Gueudin, F. Damond, A. 306
Malmsten, G.E. Corrigan, and F. Simon. 2003. A new quantitative HIV load assay 307
based on plasma virion reverse transcriptase activity for the different types, groups and 308
subtypes. AIDS. 17:331-336. 309
on April 25, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
17
4. Colebunders, R., K.R. Moses, J. Laurence, H.M. Shihab, F. Semitala, F. Lutwama, 310
S. Bakeera-Kitaka, L. Lynen, L. Spacek, S.J. Reynolds, T.C. Quinn, B. Viner, and 311
H. Mayanja-Kizza. 2006. A new model to monitor the virological efficacy of 312
antiretroviral treatment in resource-poor countries. Lancet Infect. Dis. 6:53-59. 313
5. Iqbal, H.S., P. Balakrishnan, A.J. Cecelia, S. Solomon, N. Kumarasamy, V. 314
Madhavan, K.G. Murugavel, A.K. Ganesh, S.S. Solomon, K.H. Mayer, and S.M. 315
Crowe. 2007. Use of an HIV-1 reverse-transcriptase enzyme-activity assay to measure 316
HIV-1 viral load as a potential alternative to nucleic acid-based assay for monitoring 317
antiretroviral therapy in resource-limited settings. J. Med. Microbiol. 56:1611-1614. 318
6. Garcia-Diaz, A., G.S. Clewley, C.L. Booth, W. Labett, N. McAllister, and A.M. 319
Geretti. 2006. Comparative evaluation of the performance of the Abbott real-time 320
human immunodeficiency virus type 1 (HIV-1) assay for measurement of HIV-1 321
plasma viral load following automated specimen preparation. J. Clin. Microbiol. 44: 322
1788-1791. 323
7. Geretti AM. Epidemiology of antiretroviral drug resistance in drug-naïve persons. 2007. 324
Curr Opin Infect Dis. 20:22-32. 325
8. Goldie, S.J., Y. Yazdanpanah, E. Losina, M.C. Weinstein, X. Anglaret, R.P 326
Walensky, H.E. Hsu, A. Kimmel, C. Holmes, J.E. Kaplan, and K.A. Freedberg. 327
2006. Cost-effectiveness of HIV treatment in resource-poor settings-the case of Cote 328
d’Ivoire. N. Engl. J. Med. 355:1141-1153. 329
9. Greengrass, V., M. Plate, P. Steele, J. Denholm, C.L. Cherry, L. Morris and S.M. 330
Crowe. 2008. Evaluation of the new version 3 Cavidi ExaVir™ Load quantitative HIV 331
RT load kit as an alternative HIV viral load monitoring assay for use in Melbourne, 332
on April 25, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
18
Australia. XVII International AIDS Conference, 3-8 August 2008, Mexico City, 333
Abstract Number THPE0054. 334
10. Gueudin, M., J.C. Plantier, V. Lemee, M.P. Schmitt, L. Chartier, T. Bourlet, A. 335
Ruffault, F. Damond, M. Vray, and F. Simon. 2007. Evaluation of the Roche Cobas 336
TaqMan and Abbott RealTime extraction-quantification systems for HIV-1 subtypes. J. 337
Acquir. Immune Defic. Syndr. 44:500-505. 338
11. Hughes, M.D., V.A. Johnson, M.S. Hirsch, J.W. Bremer, T. Elbeik, A. Erice, D.R. 339
Kuritzkes, W.A. Scott, S.A. Spector, N. Basgoz, M.A. Fischl, and R.T. D'Aquila. 340
1997. Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count 341
improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol 342
Virology Substudy Team. Ann Intern Med.126:929-938. 343
12. Jennings, C., S.A. Fiscus, S.M. Crowe, A.D. Danilovic, R.J. Morack, S. Scianna, A. 344
Cachafeiro, D.J. Brambilla, J. Schupbach, W. Stevens, R. Respess, O.E. Varnier, 345
G.E. Corrigan, J.S. Gronowitz, M.A. Ussery, and J.W. Bremer. 2005. Comparison 346
of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard 347
HIV RNA assay. J. Clin. Microbiol. 43:5950-5956. 348
13. Kumarasamy, N., S. Solomon, S.K. Chaguturu, A.J. Cecelia, S. Vallabhaneni, T.P. 349
Flanigan, and K.H. Mayer. 2005. The changing natural history of HIV disease: before 350
and after the introduction of generic antiretroviral therapy in southern India. Clin. Infec. 351
Dis. 41:1525-1528. 352
14. Lombart, J.P., M. Vray, A. Kafando, V. Lemée, R. Ouédraogo-Traoré, G.E. 353
Corrigan, J.C. Plantier, F. Simon, and J. Braun. 2005. Plasma virion reverse 354
on April 25, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
19
transcriptase activity and heat dissociation-boosted p24 antigen assay for HIV load in 355
Burkina Faso, West Africa. AIDS. 19:1273-1277. 356
15. Malmsten, A., X.W. Shao, S. Sjödahl, E.l. Fredriksson, I. Pettersson, T. Leitner, 357
C.F. Källander, E. Sandström, J.S. Gronowitz. 2005. Improved HIV-1 viral load 358
determination based on reverse transcriptase activity recovered from human plasma. J. 359
Med. Virol. 76:291-296. 360
16. Manavi, K. 2008. The significance of low level plasma HIV viral load on COBAS 361
TaqMan HIV-1 assay for patients with undetectable plasma viral load on COBAS 362
Amplicor Monitor version 1.5. HIV Clin. Trials. 9:283-286. 363
17. Moore, D.M., A. Awor, R. Downing, J. Kaplan, J.S. Montaner, J. Hancock, W. 364
Were, and J. Mermin J. 2008. CD4+ T-cell count monitoring does not accurately 365
identify HIV-infected adults with virologic failure receiving antiretroviral therapy. J 366
Acquir Immune Defic Syndr. 49:477-4484. 367
18. Murri, R., A.C. Lepri, P. Cicconi, A. Poggio, M. Arlotti, G. Tositti, D. Santoro, 368
M.L. Soranzo, G. Rizzardini, V. Colangeli, M. Montroni, and A.D. Monforte. 369
2006. Is moderate HIV viremia associated with a higher risk of clinical progression in 370
HIV-infected people treated with highly active antiretroviral therapy: evidence from the 371
Italian cohort of antiretroviral-naïve patients study. J. Acquir. Immune Defic. Syndr. 372
41:23-30. 373
19. Phillips, A.N., D. Pillay, A.H. Miners, D.E. Bennett, C.F. Gilks, and J.D. 374
Lundgren. 2008. Outcomes from monitoring patients on antiretroviral therapy in 375
resource-limited settings with viral load, CD4 cell count or clinical observation alone: a 376
computer simulation model. Lancet. 371:1443-1451. 377
on April 25, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
20
20. Reynolds, S.J., G. Nakigozi, K. Newell, A. Ndyanabo, R. Galiwongo, I. Boaz, T.C. 378
Quinn, R. Gray, M. Wawer, and D. Serwadda. 2009. Failure of immunologic criteria 379
to appropriately identify antiretroviral treatment failure in Uganda. AIDS. 23:697-700. 380
21. Schutten, M., D. Peters, N.K.T. Back, M. Beld, K. Beuselinck, V. Foulongne, A.M. 381
Geretti, L. Pandiani, C. Tiemann, and H.G. Niesters. 2007. Multicenter evaluation 382
of the new Abbott RealTime assay for quantitative detection of Human 383
Immunodeficiency Virus type 1 and Hepatitis C Virus RNA. J. Clin. Microb. 45:1712-384
1717. 385
22. Severe, P., P. Leger, M. Charles, F. Noel, G. Bonhomme, G. Bois, E. George, S. 386
Kenel-Pierre, P.F. Wright, R. Gulick, W.D. Johnson Jr, J.W. Pape, and D.W. 387
Fitzgerald. 2005. Antiretroviral therapy in a thousand patients with AIDS in Haiti. N. 388
Engl. J. Med. 353:2325-2334. 389
23. Sivapalasingam, S., S. Essajee, P.N. Nyambi, V. Itri, B. Hanna, R. Holzman, and F. 390
Valentine. 2005. Human immunodeficiency virus (HIV) reverse transcriptase activity 391
correlates with HIV RNA load: implications for resource-limited settings. J. Clin. 392
Microbiol. 43:3793-3796. 393
24. Steegen, K., S. Luchters, N. De Cabooter, J. Reynaerts, K. Mandaliya, J. Plum, W. 394
Jaoko, C. Verhofstede, and M. Temmerman. 2007. Evaluation of two commercially 395
available alternatives for HIV-1 viral load testing in resource-limited settings. J Virol 396
Methods. 146:178-187. 397
25. Stevens, G., N. Rekhviashvili, L.E. Scott, R. Gonin, and W. Stevens. 2005. 398
Evaluation of two commercially available, inexpensive alternative assays used for 399
on April 25, 2020 by guest
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assessing viral load in a cohort of human immunodeficiency virus type 1 subtype C-400
infected patients from South Africa. J. Clin. Microbiol. 43:857-861. 401
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26. Table 1. Comparison of HIV-1 plasma viral load levels measured by the Cavidi 402
ExaVirTM Load v.3 assay, the Abbott M2000sp/M2000rt RealTime HIV-1 assay and the 403
Roche COBAS Ampliprep/COBAS TaqMan HIV-1 v.1 assay, according to the 404
antiretroviral (ARV) treatment status and HIV-1 subtype. The mean and standard 405
deviation (SD) are shown, calculated after conversion into log10 copies/ml. 406
Undetectable viral load levels were scored as 1.6 log10 (40) copies/ml, corresponding to 407
the lower limit of quantification of the two molecular assays. 408
Mean viral load Log10 copies/ml (SD)
Characteristics
Number
ExaVir RealTime TaqMan
All 119 3.3 (1.4) 3.6 (1.4) 3.5 (1.4)
Naïve 71 4.2 (0.9) 4.5 (0.9) 4.3 (0.9) ARV status
Experienced 48 2.1 (0.9) 2.3 (1.0) 2.3 (1.0)
A 19 3.0 (1.4) 3.2 (1.5) 3.2 (1.4)
B 34 3.5 (1.3) 3.8 (1.3) 3.7 (1.2)
C 30 3.0 (1.3) 3.3 (1.4) 3.3 (1.3)
D 9 3.7 (1.2) 3.8 (1.3) 3.8 (1.2)
F 2 2.2 (0.6) 2.5 (0.9) 2.5 (0.9)
G 1 3.3 3.8 3.0
H 1 5.1 5.6 5.5
CRF01 3 2.4 (1.1) 2.2 (0.7) 1.8 (0.2)
CRF02 13 4.2 (1.2) 5.0 (0.9) 4.6 (0.9)
CRF06 1 1.6 2.0 1.8
CRF12 1 1.6 2.0 1.9
CRF14 1 5.5 5.7 4.0
Subtype
Cpx 4 2.4 (1.3) 2.7 (1.3) 2.7 (1.4)
409
Cpx= complex mosaic pol sequence 410
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Table 2. Samples showing HIV-1 plasma viral load levels outside the 95% level of 411
agreement between two assays when tested by the Cavidi ExaVirTM Load v.3 assay, the 412
Abbott M2000sp/M2000rt RealTime HIV-1 assay and the Roche COBAS 413
Ampliprep/COBAS TaqMan HIV-1 v.1 assay. 414
415
416
417
418
419
420
421
422
Viral load (log10 copies/ml) Subtype
ExaVir RealTime TaqMan
A Undetectable 3.3 2.4
B 3.8 5.2 5.0
B 4.8 5.2 4.2
D Undetectable 3.0 3.0
D 3.1 <1.6 <1.6
D 4.4 4.8 3.7
D 3.6 3.0 4.0
CRF01 3.9 3.2 <1.6
CRF02 3.3 5.5 5.2
CRF02 2.7 4.3 3.7
CRF02 Undetectable 3.0 3.2
CRF02 5.4 5.7 4.7
CRF14 5.5 5.7 4.0
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Figure 1. Correlation between the Cavidi ExaVirTM Load v.3 assay and the Abbott 423
M2000sp/M2000rt RealTime HIV-1 assay (a), ExaVir and the Roche COBAS 424
Ampliprep/COBAS TaqMan HIV-1 v.1 assay (b), and RealTime and TaqMan (c), 425
determined by parallel testing of 119 samples. All viral load values are in log10 copies/ml. 426
Undetectable viral load levels were scored as 1.6 log10 (40) copies/ml, corresponding to the 427
lower limit of quantification of the two molecular assays. The linear regression line is 428
shown. 429
430
Figure 2. Bland-Altman analysis of the agreement between the Cavidi ExaVirTM Load v.3 431
assay and the Abbott M2000sp/M2000rt RealTime HIV-1 assay (a), ExaVir and the Roche 432
COBAS Ampliprep/COBAS TaqMan HIV-1 v.1 assay (b), and RealTime and TaqMan (c). 433
The labels show the subtype of samples outside the 95% level of agreement, given by the 434
mean difference plus or minus twice the standard deviation of the difference. 435
436
Figure 3. Comparison of viral load measurements obtained by by the Cavidi ExaVirTM 437
Load v.3 assay, the Abbott M2000sp/M2000rt RealTime HIV-1 assay and the Roche 438
COBAS Ampliprep/COBAS TaqMan HIV-1 v.1 assay with serial dilutions of the WHO 2nd 439
International Standard for HIV-1 RNA ranging from 4.4 to 1.6 log10 (25,000 to 40) IU/ml. 440
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0.0
1.0
2.0
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