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A new sensitive one-step real-time duplex PCR method for groups A and B HIV-1 2 RNA load 2 3 Véronique Avettand-Fenoel#1,2, Florence Damond#3, Marie Gueudin4, Sophie 4 Matheron5, Adeline Mélard2, Gilles Collin3, Diane Descamps3, Marie-Laure Chaix6, 5 Christine Rouzioux1,2, and Jean-Christophe Plantier4 for the ANRS-CO5 HIV-2 and the 6 ANRS-AC11 working group. 7 #: equal contribution 8 1- Laboratoire de Virologie, AP-HP, Hôpital Necker Enfants malades, Paris, France 9 2- Université Paris-Descartes, Sorbonne Paris Cité, Faculté de Médecine, EA7327, Paris, France 10 3- INSERM, IAME, UMR 1137, Paris, France; Université Paris Diderot, Sorbonne Paris Cité, Paris, 11 France; AP-HP, Hôpital Bichat-Claude Bernard, Laboratoire de Virologie, Paris, France 12 4- Laboratoire associé au Centre National de Référence du VIH, hôpital Charles Nicolle, CHU de 13 Rouen, Rouen, France; GRAM, Equipe d’Accueil 2656, Faculté de Médecine-Pharmacie, Institut de 14 Recherche et d'Innovation en Biomédecine, Université de Rouen, Rouen, France. 15 5- INSERM, IAME, UMR 1137, F-75018 Paris, France; Université Paris Diderot, Sorbonne Paris Cité, 16 F-75018 Paris, France; AP-HP, Hôpital Bichat-Claude Bernard, Service de Maladies Infectieuses et 17 Tropicales, F-75018 Paris, France 18 6- Laboratoire de Virologie, AP-HP, Hôpital Saint-Louis, Paris, France 19 20 Key words 21 HIV-2; viral load; genetic diversity; duplex RT-PCR 22 23 Corresponding author: Pr J.C. Plantier 24 Laboratoire de Virologie, Institut de Biologie Clinique, 25 Hôpital Charles Nicolle, CHU de Rouen, 26 1 rue de Germont, 76031, Rouen, 27 France; Tel: + 33 2 32 88 14 62 Fax: + 33 2 32 88 04 30 28 Electronic address: Jean-Christophe.Plantier@univ-rouen.fr 29

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JCM Accepts, published online ahead of print on 11 June 2014J. Clin. Microbiol. doi:10.1128/JCM.00724-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 31 The French National Agency for HIV/AIDS Research has previously developed a widely 32 used method for HIV-1 RNA quantification (Biocentric). Here we developed a new specific 33 and sensitive method for HIV-2 RNA quantification, based on an adaptation of the 34 existing HIV-1 protocol. 35 The new test is based on Taqman one-step qRT-PCR targeting two conserved consensus 36 regions of HIV-2 (LTR and Gag). Analytic performances were determined in three 37 laboratories. Clinical performances were evaluated on 100 plasma samples from HIV-2-38 infected patients (group A, B and H), by comparison with the assay currently used for the 39 ANRS HIV-2 cohort. 40 The specificity was 100%. Sensitivity was 50 cp/mL and could be optimized to 10 cp/mL. 41 The within-run coefficients of variation in the three laboratories varied from 0.54% to 42 1.61% at 4 log10 copies/mL, and from 7.24% to 14.32% at 2 log10 cp/mL. The between-43 run coefficients of variation varied from 2.28 to 6.43%. 44 Of the 39 clinical samples below 2 log10 in the current assay, the new test improved the 45 detection or quantification of 17 samples, including eight group B. For quantifiable 46 samples, similar loads were obtained with the two assays for group A samples. The 47 median difference between the two assays for group B samples was +0.18, but with 48 greater heterogeneity. The HIV-2 group H sample gave similar results with the two 49 assays. 50 This new assay is highly sensitive and accurately quantifies the most prevalent HIV-2 51 groups. This test will be useful for monitoring low loads in HIV-2 infected patients. 52

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Introduction 54 HIV-2 is characterized by less efficient transmission through the sexual and vertical routes 55 than HIV-1 (2, 15) , and by a slower natural clinical course (15, 16, 30) ; nevertheless, 56 HIV-2 infection eventually leads to AIDS. HIV-2 infection must be distinguished from HIV-57 1 infection, as HIV-2 is naturally resistant to non nucleoside reverse transcriptase 58 inhibitors, T20, and some protease inhibitors, and as patient follow-up differs from that of 59 HIV-1 infection (12, 31). 60 Compared to HIV-1, HIV-2 is characterized by lower viral replication (17-19, 21, 30) . In 61 the French ANRS cohort of HIV-2-infected patients (974 patients in June 2013), 61% of 62 untreated patients have plasma viral loads below 250 copies/mL (cp/mL). Likewise, in a 63 British study, only 8% of patients with CD4 >500 cells/mm3 and 62% of patients with 64 CD4 <300 cells/mm3 had detectable viral load (27), implying that 38% of patients had 65 undetectable viral load in an assay with a quantification limit of 100 copies/ml. 66 Clinical management of HIV-2 infection is hampered by the lack of validated commercial 67 RNA viral load assays. In-house assays are therefore widely used (5, 9, 11, 13, 22, 25, 68 29). The ACHIEV2E international collaboration on HIV-2 infection showed that plasma 69 HIV-2 RNA values vary considerably between laboratories (7,8). The high genetic 70 diversity of HIV-2, with 9 groups designated A to I, of which only groups A and B are 71 epidemic, also represents an obstacle to accurate viral load quantification (3, 10, 14, 26, 72 28) : previous studies show that group B viruses are particularly difficult to quantify (7-73 9). 74 As most cases of HIV-2 infection occur in resource-limited settings, an affordable test is 75 urgently needed, particularly to identify HIV-1/HIV-2 coinfection. The French national 76 agency for HIV/AIDS research (ANRS) has previously promoted the development of an 77 HIV-1 RNA assay, which is now marketed by Biocentric (Bandol, France) and is widely 78 used in resource-limited settings (1, 23). This test is easy to perform and affordable, and 79 has been proven useful for patient monitoring. 80 The aim of the present work was to develop a new sensitive method for quantifying HIV-81 2 RNA, particularly that of epidemic groups A and B. It is based on a one-step real-time 82

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duplex PCR method using the same amplification protocol as the Biocentric generic HIV-1 83 charge virale assay. The new test was compared with the HIV-2 assay currently used for 84 the French HIV-2 cohort and was validated by three laboratories belonging to the ANRS 85 AC11 quantification working group. 86 87 Materials and Methods 88 89 The new HIV-2 RNA assay 90 The new test is based on a one-step duplex Taqman PCR approach targeting a conserved 91 consensus region in the long terminal repeat (LTR) region and the Gag region. The 92 forward and reverse primers for the LTR region are 5’-TCTTTAAGCAAGCAAGCGTGG-3’ 93 and 5'-AGCAGGTAGAGCCTGGGTGTT-3’, respectively (24), with a new internal probe (5’ 94 FAM- CTTGGCCGGYRCTGGGCAGA-BHQ1) to optimize efficiency for HIV-2 group B. The 95 forward and reverse primers for the Gag region are F3 5’-GCGCGAGAAACTCCGTCTTG-3’ 96 and R1 5'-TTCGCTGCCCACACAATATGTT-3’, respectively (9), and the internal HIV-2 97 Taqman gag probe is S65GAG2 6 FAM-TAGGTTACGGCCCGGCGGAAAGA- BHQ13’ 98 (Eurogentec, Seraing, Belgium) (9). 99 RNA was extracted from 200 µl of plasma by using the QIAamp viral RNA mini kit 100 (Qiagen, Courtaboeuf, France), as in the Biocentric generic HIV-1 charge virale assay, in 101 laboratories A and B (Necker Hospital, Paris, and Charles Nicolle Hospital, Rouen) or 1 ml 102 with the Total NA large volume MagnaPure kit (Roche Automated System, Meylan, 103 France) in laboratory C (Bichat Claude Bernard Hospital, Paris). 104 The reaction mix consists of a 20-µL volume containing the RNA extract (10 µL), primers 105 (500 nM each), probes (250 nM each), and 1X PCR buffer (4X One-step mix, Invitrogen, 106 Cergy Pontoise, France). 107 The thermocycling conditions are those used for the Biocentric HIV-1 assay: 10 min at 108 50°C and 5 min at 95°C, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. 109 Amplification and data acquisition are carried out with the TaqMan ABI realtime PCR 110 system (Applied Biosystems, Courtaboeuf, France). The log10 number of targets initially 111

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present is proportional to the cycle threshold (CT) and is determined from the standard 112 curve. 113 A BIOQ HIV-2 RNA group A quantification panel (P0182; Rijswijk, The Netherlands) was 114 used as the external standard. The standard, evaluated at 2.93x106 cp/ml, was first 115 diluted in RPMI culture medium to a theoretical concentration of 1 000 000 cp/ml (2 116 400 000 IU/ml), followed by serial 10-fold dilution to concentrations ranging from 1 117 000 000 (5 log10) to 100 cp/ml (2 log10), and a final dilution to 40 cp/ml (1.6 log10). 118 119 Determination of the analytic performance of the new assay 120 Specificity was determined by testing plasma samples from 49 HIV-negative subjects and 121 30 HIV-1 group M-positive patients with viral loads ranging from >20 to <10 000 000 122 cp/mL. Nine HIV-1 group O coculture supernatants were also tested. 123 Linearity was assessed using the BIOQ external standard diluted in RPMI to 1 000 000, 124 100 000, 10 000, 1000, 100 and 40 cp/ml (each tested in 10 runs at Lab A). 125 Analytical sensitivity was determined by dilution in RPMI of the BIOQ external standard 126 to 100, 50, 40, 20 and 10 cp/mL (10 replicates each). 127 To determine within-run reproducibility, the BIOQ external standard was tested at 128 concentrations of 10 000 and 100 cp/mL in each of the three laboratories (10 replicates 129 for each dilution). 130 To determine between-run reproducibility, an HIV-2 positive control was prepared by 131 serial dilution in HIV-negative EDTA human plasma of a coculture supernatant of an HIV-132 2 group A isolate (Genbank accession number AY688870). This solution was diluted to 133 obtain aliquots with theoretical concentrations ranging from 10 000 to 100 000 cp/mL in 134 the current assay. These aliquots were each tested once in 7 separate runs with the 135 Magna pure automated extraction system (Lab C) and in respectively 18 and 7 separate 136 runs with Qiagen manual extraction at Lab A and Lab B. 137 138 Statistical analysis 139 MedCalc software (Ostend, Belgium) was used for data analysis. Bland and Altman 140

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curves were used to represent the degree of agreement between the two techniques (4). 141 The X-axis bore the mean values for each sample obtained with the two techniques, and 142 the Y-axis the difference between the values obtained with the two techniques. 143 Disagreement between the two techniques was defined as a difference of more than 0.5 144 log10 for a given sample. 145 146 Clinical samples 147 One hundred plasma samples from HIV-2-infected patients (n=100) included in the 148 French National HIV-2 Cohort (ANRS CO05) were selected according to the viral 149 genotype and the HIV-2 RNA concentration, as determined with our current in-house 150 technique (9). Among them, fifty-one percent were treated by antiretrovirals. The HIV-2 151 group was determined for 89 samples, as previously described (10, 20): 38 samples 152 were group A, 50 group B, and one group H. Genotyping was not available for the 153 remaining 11 samples, owing to the absence of detectable RNA and a lack of whole blood 154 or mononuclear cells for viral DNA assay. 155 The selected samples had the following characteristics: <100 (2 log10) cp/ml (n=39, 9 156 group A and 19 group B, 11 non genotypable), 100 (2 log10) - 1000 (3 log10) cp/ml 157 (n=16, 5 A and 11 B), 1000 (3 log10) - 10 000 (4 log10) (n=22, 12 A and 10 B), 10 000 158 (4 log10) - 100 000 (5 log10) (n=19, 11 A, 7 B and 1 H) and >100 000 (5 log10) (n=4, 1 A 159 and 3B). 160 161 RESULTS 162 Analytic performances of the new assay 163 As expected, given the wide genomic divergence between HIV-1 and HIV-2, the HIV-2 164 primers did not hybridize to HIV-1 genes: all HIV-1-positive plasma samples and all HIV-165 negative samples were negative in the new assay, giving a specificity of 100%. 166 The standard curve showed a strong linear relationship between the CT values and log10 167 HIV-2 RNA cp/mL (figure 1). The median correlation coefficient was 0.9947 (range, 168 0.9831 to 0.9997), and the median slope was -3.37 (range, -3.16 to -3.62). 169

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The analytical sensitivity of the assay was 100% at 40 cp/ml (1.6 log10 cp/mL) and 90% 170 at 20 cp/mL (1.3 log10 cp/mL) after Roche Magna pure automated extraction of 1 mL, 171 and 100% at 50 cp/mL (1.7 log10 cp/mL) after manual extraction of 200 µL. Optimization 172 of the assay sensitivity after manual extraction was evaluated using 1 mL of plasma: the 173 sample was centrifuged at 17 000 rpm and the pellet was resuspended in 200 µL of RPMI 174 medium prior to manual extraction as previously described, with elution in 60 µL. This 175 yielded 90% sensitivity at 10 cp/mL (1 log10 cp/mL). 176 Within-run reproducibility was evaluated in the three labs by using the BIOQ external 177 standard with theoretical virus concentrations of 10 000 and 100 cp/mL (4 and 2 log10 178 cp/mL): for the 4 log10 cp/mL value we obtained a mean of 3.91 log10 cp/mL at Lab C, 179 4.1 log10 cp/mL at Lab A and 4.2 log10 cp/mL at Lab B, with within-run coefficients of 180 variation of 1.61%, 0.54% and 1.10%, respectively. At the concentration of 2 log10 181 cp/mL, we obtained mean values of 2.03 log10 cp/mL at Lab B, 2.07 log10 cp/mL at Lab A, 182 and 2.17 log10 cp/mL at Lab C, with within-run coefficients of variation of 10.72%, 183 14.32% and 7.24%, respectively. 184 In between-run assays, the positive control with a theoretical concentration between 10 185 000 (4 log10) and 100 000 cp/mL (5 log10) was evaluated at 4.61 log10 cp/mL in Lab C, 186 4.70 log10 cp/mL in Lab A, and 4.88 log10 cp/mL in Lab B, with coefficients of variation of 187 2.28%, 6.43% and 3.03%, respectively. 188 189 Clinical performances 190 The clinical performances of the new assay were evaluated in Lab C. Clinical samples of 1 191 ml were extracted with the automated MagnaPure method and then the same elute was 192 used to perform the two assays in parallel with the ABI device for the new assay and the 193 Light Cycler 1.5 device for the current assay. The results obtained with the new assay 194 were categorized into four groups (table 1): undetectable (<40 cp/mL), detectable but 195 not quantifiable (0 to <40 cp/mL), quantifiable between 40 and 100 cp/mL, and above 196 the lower limit of quantification of the current assay (100 cp/mL). 197 Of the 39 samples below the quantification limit of 100 cp/mL in the current assay, 22 198

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samples (56%) were also undetectable with the new assay (table 1), while 10 samples 199 (26%; 3 A, 3 B and 4 non genotypable) were detected at values between 0 and 40 200 cp/mL (range: 1 to 36 cp/mL). Three samples (7.7%; 1 B, 2 non genotypable) were 201 quantified between 40 and 100 cp/mL (range: 56 to 79 cp/mL), and four samples (10%; 202 all B) were quantified above 100 cp/mL (range: 102 to 970 cp/mL); the latter 203 corresponded to true false-negative samples, taking into account the 100 cp/mL cut-off 204 of the current assay. These results showed that the new test improved the detection or 205 quantification of 17/39 samples (43.6%), including eight group B samples (table 1). 206 All 61 plasma samples with values above 100 cp/mL in the current assay were detectable 207 with the new test. One sample at 209 cp/mL (2.32 log10 cp/ml) in the current assay gave 208 a value of 46 cp/mL (1.69 log10 cp/ml) in the new test (table 1). 209 A scatter plot was constructed with the values obtained for the 78 samples detectable or 210 quantifiable with the new assay (figure 2). It showed a wider dispersion of values for 211 quantifiable group B samples than for quantifiable group A samples, as well as better 212 detection or quantification of group B and non genotypable samples. This was confirmed 213 by scatter equations specific for group A samples (n=32; y = 0.9485x + 0.0294, 214 r2=0.9619) and group B samples (n=39; y = 0.7766x + 0.8087, r2=0,8184) and also by 215 Bland-Altman representations (figure 3). Homogeneous quantification (+/-1.96 SD, 216 range from –0.6 to 0.35) and similar values (median difference of -0.13) were obtained 217 with the new and current assays for group A samples. The median difference between 218 the two assays for group B samples was +0.18, but with greater heterogeneity (+/-1.96 219 SD, range –0.98 to 1.33). 220 Among the samples that were quantifiable with both assays, 10 samples showed 221 differences above 0.5 log10, nine of them belonging to group B. Five of these samples 222 were better quantified with the new assay (differences of 0.53, 0.60, 0.68, 0.76 and 0.89 223 log10/mL) and five with the current assay (differences of 0.51, 0.52, 0.55, 0.66 and 0.70 224 log10/mL). These differences illustrate the difficulty of correctly assessing HIV-2 group B 225 viral load. 226 The only HIV-2 group H sample gave very similar results with the two assays (4.33 log10 227

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and 4.34 log10). 228 229

Discussion 230 HIV-2 infection differs markedly from HIV-1 infection, notably by its slower natural 231 course, different therapeutic management, and genetic diversity. Specific molecular 232 methods are therefore necessary for diagnosis and patient monitoring. Current assays, 233 mainly consisting of in-house methods or unvalidated derivatives of commercial kits, 234 suffer from major limitations in terms of their sensitivity, accuracy, and coverage of HIV-235 2 genetic diversity (7, 8). 236 The aim of this work was to develop a quantitative assay that takes into account both the 237 low viral load seen in most HIV-2-infected patients and the broad genetic diversity of 238 HIV-2, especially group B (7-9). In addition, as most cases of HIV-2 infection occur in 239 West Africa, such a test must be easy to implement in developing countries, as 240 previously achieved with the generic HIV-1 viral load assay marketed by Biocentric. 241 We chose to optimize the assay currently used to monitor the French HIV-2 cohort based 242 on amplification of the HIV-2 Gag region, that gave accurate results in the ACHIEV2E 243 study (7, 8), but with a lower limit of quantification of 100 cp/ml (9) and not completely 244 adapted to some group B strains. We also used the same operating conditions as those of 245 the Biocentric HIV-1 assay kit, in order to facilitate its use either for HIV-2 alone or 246 jointly for HIV-1 and HIV-2. 247 The new test exhibits good linearity (40 to 1 000 000 cp/ml) and within-run 248 reproducibility (<15%). Its inter-laboratory reproducibility was validated by evaluation at 249 three different sites. Both manual and automated extraction methods were validated, for 250 compatibility with local practices in resource-limited countries. 251 Relative to the current assay, the new test has a significantly better analytical limit of 252 quantification, reaching 50 cp/ml with manual extraction of 200 µl of plasma and 40 253 cp/ml with automated extraction of 1 mL. Assuming a probit rate of 90%, the detection 254 limit with 1 ml of plasma would be 10 cp/ml and 20 cp/ml, respectively. This very good 255 analytical sensitivity matches that of recently published in-house methods (5, 11, 29) 256

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and is compatible with virological monitoring of HIV-2 infection, as more than 60% of 257 untreated patients have viral loads below 250 cp/ml. The new test was able to detect 258 and/or quantify more than one-third of samples that were undetectable with our current 259 assay, which has a quantification limit of 100 cp/ml. This excellent sensitivity should 260 prove useful both for pathophysiological studies and for treatment monitoring. 261 262 The most difficult issue facing the development of HIV-2 viral load assays is the genetic 263 diversity of this virus (especially group B), some variants being under-quantified or 264 escaping detection with current tests (7-9). Three teams recently reported improved 265 sensitivity for HIV-2, but they mainly used supernatants (11) or a limited number of 266 samples (5, 29) or validated detection but not quantification (29), leaving questions as 267 to their clinical performance, especially for group B viruses. We evaluated our new assay 268 on 100 clinical samples, 39% of which were undetectable with our current assay, 269 representative of the molecular epidemiology of groups A and B, plus the only one 270 divergent sample of group H. Half the samples corresponded to group B, and more than 271 one-third of them (n=19) were undetectable with our current assay. We chose to develop 272 a duplex method capable of simultaneously amplifying the LTR and Gag regions, in order 273 to retain the benefits of each previous test and to improve the detection of group B 274 viruses by reducing the risk of mismatches. Our strategy based on coupling of primers 275 and probes in two distinct regions of the viral genome has already been adopted by 276 Roche in version 2.0 of its CAP CTM HIV1 assay, resulting in better quantification of HIV-277 1 non B subtypes (6, 41). The new and current HIV-2 assay methods gave similar results 278 for the single group H sample and for the group A samples (although 3 additional group 279 A samples were detectable with the new test), whereas the new test developed by 280 Delarue et al. gave values nearly 0.5 log10 lower than their reference test (11). Eight 281 additional group B samples (42%) were detected or quantified with our new test, four 282 samples having values of 102 to 970 cp/mL. This improvement is due to the addition of 283 primers in the LTR region and to changes in the LTR probe (data not shown). However, 284 the wider dispersion of values and the larger number of group B than group A samples 285

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with differences exceeding 0.5 log10 relative to the current assay illustrate the greater 286 difficulty of group B quantification. In addition, six non-genotypable samples were better 287 detected or quantified with our new assay. 288 289 We designed the new test for use in the same operating conditions as the generic HIV-1 290 RNA assay currently used with success in many resource-limited countries (1, 23) 291 meaning it can be used on the same machine, with the same software program and 292 even, if necessary, in the same amplification plate, as HIV-1 samples. This will reduce 293 analytical costs by increasing the number of samples per run. 294 295 Although ours is an in-house assay, its performance is adequate for patient monitoring in 296 the absence of a validated commercial test. In addition, the use of a standardized kit that 297 includes an external standard will improve inter-laboratory reliability, as shown by the 298 2nd International ACHIEV2E study (7, 8). 299 Our new assay has similar analytical performances to that of other newly developed tests 300 (5, 11, 29). However, it is difficult to compare our method with the test described by 301 Chang et al., adapted from the Abbott platform (Abbott Molecular, Chicago, Il), as the 302 latter was evaluated on few group B samples and was not compared with other 303 techniques (5). Styer et al. recently compared their method with this "Abbott" technique 304 and observed a difference of -0.35 log10 UI/mL, but they used a limited panel of 305 uncharacterized samples, ruling out any evaluation of in terms of genetic diversity (29). 306 Finally, Styer and Delarue used a two-step method, whereas our assay is performed in a 307 single step (11, 29). 308 309

In conclusion, we have developed and standardized an assay with better analytical 310 sensitivity than the technique currently used to monitor HIV-2-infected patients in 311 France. Our assay also has improved clinical sensitivity and has been validated on a 312 broad, well-characterized sample panel, contrary to recently published tests (5, 11, 29)). 313 The analytical performance of this new assay, which is easy to perform, makes it suitable 314

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for use in resource-limited countries in which multiple HIV-2 variants circulate. In 315 addition, our assay can be used on the same analytical platforms and in the same run as 316 tests for HIV-1, thus improving its cost-efficiency for monitoring patients infected with 317 HIV-1 and/or HIV-2. This possibility of simultaneous analysis will facilitate molecular 318 diagnosis of mother-to-child transmission of HIV-1 and/or HIV-2, and also diagnosis and 319 follow-up of dual HIV-1/HIV-2 infection in the same sample. Finally, use of this assay for 320 virological monitoring will provide new insights into the natural history of HIV-2 infection 321 at different clinical stages. 322

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Acknowledgments: this work was supported by the French national agency for AIDS 323 research (ANRS). We also thank the HIV-2 cohort (ANRS CO5), which is supported by a 324 grant from ANRS (Agence nationale de recherches sur le sida et les hepatites virales). 325 326

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328

329 330 331 332 333 334 335 336 337 338 339 340 Figure 1: Standard curve of the HIV-2 RNA real-time viral load assay. The cycle threshold 341 (CT) is the number of cycles at which fluorescence passes a fixed limit (time to positivity). 342 Median values and 25% and 75% interquartile ranges (box plot) of the CT are indicated. 343 The vertical lines show the ranges of the CT. 344

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345 346 347 348

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,00,0

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Figure 2. Scatter diagram according to the genetic group of the 78 samples detectable (0 to 351 <40 cp/mL) or quantifiable (≥ 40 cp/ml) with the new assay (r2=0.8886). An arbitrary 352 value of 20 cp/ml (1.3 Log) was attributed to samples undetectable in the current assay, 353 and to those detectable in the new assay (0 to <40 cp/mL). NA: non genotypable samples. 354 355

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356 357 358 359 360 361 362 363 364 365 366 367 368 369 370

<100 cp/mL (2 Log) N=39

>100 cp/mL (2 Log) N=61

group groupA B NA A B H

VL with new version of HIV-2 assay N=9 N=19 N=11 N=29 N=31 N=1

<40 cp/mL (1.7 Log) 6 11 5

detected between 0 and 40 cp/mL 3 3 4

40 < VL <100 cp/mL 1 2 1

>100 cp/mL (2 Log) 4 28 31 1

NA: genotype not available

Table 1. results of the clinical performances of the new assay using automated extraction (MagnaPure + ABI prism, cut-off of 40 cp/mL), with those obtained with the current assay ((MagnaPure + Light Cycler method (cut-off of 100 cp/mL)

VL with current version of HIV-2 assay

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371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 Figure 3: Degree of agreement in Log10 cp/ml between the new and current assays; Bland 394 and Altman curves for group A (1; N=32) and group B (2; N=39) samples detectable (0 to 395 40 cp/mL) and quantifiable (>40 cp/mL) with the new assay. An arbitrary value of 10 cp/ml 396 was attributed to samples undetectable with the current assay, and 20 cp/ml to those 397 detectable with the new assay (between 0 and 40 cp/mL). 398 399

HIV-2 group B (39 samples)

1 2 3 4 5 6 7-1.5

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