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: a.geretti@medsch.ucl.ac.uk 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

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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

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assessing viral load in a cohort of human immunodeficiency virus type 1 subtype C-400

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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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