Comparative Evaluation of the ExaVir Load Version 3 Reverse Transcriptase Assay for Measurement of Human Immunodeficiency Virus Type 1 Plasma Load

0095-1137/09/$08.00⫹0 doi:10.1128/JCM.00715-09

Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Comparative Evaluation of the ExaVir Load Version 3 Reverse

Transcriptase Assay for Measurement of Human

Immunodeficiency Virus Type 1 Plasma Load

䌤

Wendy Labbett,

Ana Garcia-Diaz,

Zoe Fox,

Gillian S. Clewley,

Thomas Fernandez,

Margaret Johnson,

and Anna Maria Geretti

*

Department of Virology,1_{Department of Infection and Population Health,}2_{and Department of HIV Medicine,}3

Royal Free Hampstead NHS Trust and University College London Medical School, London, United Kingdom

Received 7 April 2009/Returned for modification 22 May 2009/Accepted 19 July 2009

In resource-limited settings, the virological monitoring of antiretroviral therapy is limited by high cost and the lack of infrastructure. The Cavidi ExaVir Load assay employs a simple and inexpensive enzyme-linked immunosorbent assay format to measure human immunodeficiency virus (HIV) reverse transcriptase activity, which correlates with plasma RNA load. The version 3 assay has been described as having improved precision and sensitivity. There are limited data on its performance relative to those of current real-time assays. Our objective was to compare HIV type 1 (HIV-1) RNA load measurement in plasma by ExaVir Load version 3 (designated ExaVir), Abbott M2000sp/M2000rt RealTime HIV-1 assay (designated RealTime), and Roche COBAS Ampliprep/COBAS TaqMan HIV-1 version 1 assay (designated TaqMan). Plasma from 119 patients (34 with subtype B infection, 85 with non-subtype B infection [A-H, CRF01, CRF02, CRF06, CRF12, CRF14, and complex]; 48 subjects were treatment experienced, 71 were naive) and serial dilutions of the second international standard (IS) were tested. Assay relationship and agreement were determined by linear regres-sion, correlation analysis, and the Bland-Altman method. The ExaVir assay quantified 77/83 (92.8%) samples

with viral loads of >2.3 log10copies/ml by the molecular assays. Results were linearly associated and strongly

correlated with RealTime and TaqMan measurements (Rof 0.94 and 0.92, respectively) for both subtype B (R

of 0.97 and 0.95, respectively) and non-subtype B (Rof 0.93 and 0.91, respectively) samples. Mean differences

were 0.28 and 0.18 log10copies/ml in favor of the two molecular assays; 7/119 (5.9%) and 5/119 (4.2%) samples

were outside the 95% level of agreement. ExaVir underquantified the IS by a mean of 0.2 (range, 0.0 to 0.5) log10

copies/ml. The ExaVir assay showed excellent concordance with real-time molecular assays, offering a suitable option for virological monitoring in settings with limited infrastructure.

The introduction of combination antiretroviral therapy in resource-limited countries has resulted in significant reduc-tions in morbidity and mortality (8, 13, 22). It generally is accepted that the unavailability of plasma viral load (VL) mon-itoring should not preclude expanded access to treatment in these settings (19). However, knowledge accrued through more than a decade of experience in high-income settings indicates that virological monitoring is required in treated pa-tients to ensure optimal long-term outcomes (2, 11). Incom-plete VL suppression during therapy leads to the emergence and evolution of drug resistance, reducing treatment options and resulting in the transmission of resistant mutants (7). Nei-ther clinical findings nor CD4 cell counts are adequate predic-tors of viral suppression, and in fact management by CD4 cell counts alone can lead to unnecessary treatment changes (1). VL testing is the only reliable marker for the early detection of the failure of antiretroviral therapy (17, 20).

Molecular VL assays in routine use in high-income countries require expensive instruments and reagents, sophisticated lab-oratory facilities to minimize the risk of contamination, a

reg-ular and stable electricity supply, and highly skilled laboratory technicians proficient in molecular biology techniques. These factors limit the implementation of VL testing in resource-limited settings. The Cavidi ExaVir Load assay employs a mod-ified enzyme-linked immunosorbent assay (ELISA) format to measure the viral reverse transcriptase (RT) activity, which in turn correlates with plasma RNA levels (3, 23). The assay requires simple, routinely available equipment (e.g., an incu-bator, ELISA plate reader, freezer, mixing table, and vortex machine) and is relatively inexpensive and simple to perform. The price per test is dependent on volumes but can be as low as $13.66 (M. Eales, personal communication). These charac-teristics make it suitable for use in settings with limited infra-structure. In November 2007, the manufacturer signed an agreement with the Clinton Foundation HIV/AIDS Initiative (CHAI) to provide the assay at a discounted price to members of the CHAI Procurement Consortium of more than 70 devel-oping countries. The two previous versions of the assay have been evaluated in the literature (3, 5, 12, 14, 15, 23, 25). Relative to version 2, version 3 is described by the manufac-turers as showing enhanced precision, analytical specificity and sensitivity (the lower limit of quantification was reduced from 400 to 200 copies/ml), improved turn-around time (from 72 to 48 h), reduced hands-on time (from 6 to 5 h), a reduction in the use of consumables, and increased throughput (from 120 to 180 samples per week per scientist). There is no published

* Corresponding author. Mailing address: Department of Virology, Royal Free Hampstead NHS Trust & UCL Medical School, Pond St., London NW3 2QG, United Kingdom. Phone: 44 20 7317 7521. Fax: 44 20 7830 2854. E-mail: [email protected].

䌤_{Published ahead of print on 5 August 2009.}

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evidence on the performance of the version 3 assay relative to current real-time molecular methods in use in high-income countries.

The objective of this study was to evaluate the performance of the ExaVir Load version 3 assay (referred to hereafter as the ExaVir assay) in comparison to two real-time PCR assays widely used in high-income countries: the Abbott M2000sp/ M2000rt RealTime human immunodeficiency virus type 1 (HIV-1) assay (referred to as the RealTime assay) and the Roche COBAS-Ampliprep/COBAS-TaqMan HIV-1 version 1 assay (referred to as the TaqMan assay).

(This work was presented at the 16th Conference on Retro-viruses and Opportunistic Infections, Montreal, Canada, 8 to 11 February 2009.)

MATERIALS AND METHODS

Patients and samples.Blood samples anticoagulated with EDTA were col-lected from 119 patients attending the Royal Free Hampstead NHS Trust for routine HIV care. The study population was infected with diverse HIV-1 sub-types and comprised 71 antiretroviral treatment-naive patients and 48 patients receiving antiretroviral therapy. Plasma was separated within 6 h of collection

and stored at⫺80°C in three separate aliquots prior to parallel testing in the

three assays. Serial dilutions (n⫽10) ranging from 25,000 to 40 (4.4 to 1.6 log10)

IU/ml of the WHO subtype B second international standard for HIV-1 RNA (IS) (product code 97/650; National Institute for Biological Standards and Control, United Kingdom) also were tested in parallel. Plasma samples from 10 HIV antibody-negative patients were used as negative controls to assess ExaVir assay specificity.

ExaVir assay.The ExaVir assay (Cavidi, Sweden) measures the viral RT enzymatic activity in an ELISA format. Following the separation of virus parti-cles from 1 ml of plasma using a solid-phase extraction manifold, virus is lysed to obtain the RT enzyme and the lysate is added to an RNA template bound to the solid phase in the presence of primer and RT substrate. In the presence of RT, the enzyme synthesizes a DNA strand, which is detected by anti-bromodeoxyuri-dine monoclonal antibody conjugated to alkaline phosphatase (AP). The product is quantified by the addition of a colorimetric AP substrate. The RT activity in the sample is determined by the ExaVir Load Analyzer software through a standard curve generated by an 11-point serial dilution of a known amount of recombinant HIV-1 RT. The range of quantification, as reported by the

manu-facturers, is from approximately 200 (2.3 log10) to 600,000 (5.8 log10) copies/ml.

The upper limit varies with the reading range of the ELISA plate reader.

RealTime and TaqMan assays.The RealTime and TaqMan assays employ high-throughput, automated real-time PCR methodologies targeting conserved

regions in HIV-1integraseandgag, respectively. The RNA is extracted from 1 ml

of plasma and concentrated using magnetic particle technology in the automated Abbott M2000sp instrument and the Roche COBAS Ampliprep instrument. The detection and quantification of the amplified PCR product is accomplished within hours by monitoring the emission intensity of fluorescent reporter dyes released during the amplification process. The reported range of quantification

is 40 to 10,000,000 (1.6 to 7.0 log10) copies/ml.

HIV-1 subtyping.HIV-1 subtypes were determined frompolgene sequences using the ViroSeq system (Celera Diagnostics). Briefly, after the reverse tran-scription of plasma RNA, a 1.8-kb amplicon comprising the whole of protease and codons 1 to 335 of RT underwent population sequencing in an ABI PRISM 3100 genetic analyzer. The sequences were submitted to the NCBI and Rega HIV-1 subtyping tools, and the assignment was confirmed by phylogenetic anal-ysis with Mega 4.0 using reference sequences from the Los Alamos database (www.lanl.gov).

Statistical analysis.VL measurements were log10transformed before analysis,

and the value of the assay lower limit of quantification (LLQ) was assigned to samples with VL below this level. Pair-wise Pearson’s correlation coefficients were used to assess whether VL values determined using different assays were correlated. Since correlation coefficients do not account for the fact that one assay may provide consistently higher values than another assay, pair-wise Bland-Altman plots were used to further assess the level of agreement. These plots compare two measurement techniques by plotting the difference in VL measure-ments between any two assays against the average of the two assays. These

differences then were tested using pairedttests for each pair-wise comparison.

The Pitman’s test, based on calculating the correlation between the difference

and the mean, was used to test for a null hypothesis of equal variances given

bivariate normality. Thettest also was repeated assuming unequal variances

(and unpaired data), with similar results (not shown).

RESULTS

Patient samples.The 119 samples comprised HIV-1 group

M strains representing 34 subtype B and 85 diverse non-sub-type B strains (Table 1). At the time of sampling, 71 patients were antiretroviral treatment naive and 48 were receiving an-tiretroviral therapy.

Comparison of ExaVir and RealTime assays.Overall, 78/119

(65.5%) samples were quantified by both assays, with a median VL of 4.4 (range, 2.4 to 5.8) log₁₀copies/ml by ExaVir assay and 4.6 (range, 2.4 to 6.8) log10copies/ml by RealTime assay.

There were 25/119 (21.0%) samples with undetectable VL by both assays, all from treated patients. A further 15/119 (12.6%) samples with a median VL of 2.3 (range, 1.6 to 3.3) log₁₀ copies/ml by RealTime assay showed an undetectable VL by ExaVir assay, including seven samples that were quantified by RealTime at levels above the expected LLQ of ExaVir (2.3 log₁₀; 200 copies/ml). One sample (1/119; 0.8%) (subtype D, on antiretroviral therapy) showed a VL of 1,259 (3.1 log10)

copies/ml by ExaVir but an undetectable VL by RealTime assay. The coefficient of correlation (R) between the assays was 0.94 overall and was 0.97 and 0.93 for subtype B and non-subtype B, respectively (Fig. 1a). The VL measurements dif-fered on average by 0.28 (95% confidence interval [CI], 0.19 to 0.37) log10 copies/ml in favor of the RealTime assay (P ⬍

0.0001). Pitman’s test of the difference in variance (r) was 0.107 (P ⫽ 0.248). In the Bland-Altman comparison, the limits of agreement (the reference range for difference) were⫺0.72 to 1.27 log10copies/ml (Fig. 2a). Overall, 7/119 (5.9%) samples

TABLE 1. Comparison of HIV-1 plasma VL levels measured by the ExaVir, RealTime, and TaqMan assays by antiretroviral

(ARV) treatment status and HIV-1 subtypea

Sample type No. of

samples

Mean viral load in log10copies/ml (SD)

ExaVir RealTime TaqMan

All 119 3.3 (1.4) 3.6 (1.4) 3.5 (1.4)

ARV status

Naı¨ve 71 4.2 (0.9) 4.5 (0.9) 4.3 (0.9) Experienced 48 2.1 (0.9) 2.3 (1.0) 2.3 (1.0)

Subtype

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

Cpx 4 2.4 (1.3) 2.7 (1.3) 2.7 (1.4)

a

The means and standard deviations are shown and were calculated after

conversion into log10copies/milliliter. Undetectable VL levels were scored as 40

(1.6 log10) copies/ml, corresponding to the lower limit of quantification of the

two molecular assays. Cpx, complex mosaicpolsequence.

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(three CRF02, two subtype D, one subtype A, and one subtype B) fell outside the reference range, including six samples un-derquantified by ExaVir assay and the subtype D sample quan-tified by ExaVir but not by RealTime assay (Table 2).

Comparison of ExaVir and TaqMan assays.Overall, 77/119

(64.7%) samples were quantified by both assays, with a median VL of 4.4 (range, 2.4 to 5.8) log₁₀copies/ml by ExaVir assay and 4.5 (range, 2.8 to 6.8) log₁₀copies/ml by TaqMan assay. There were 25/119 (21.0%) samples with undetectable VL by both assays, all from treated patients. A further 15/119 (12.6%) samples with a median VL of 2.3 (range, 1.8 to 3.2) log10

copies/ml by TaqMan assay showed an undetectable VL by ExaVir assay, including seven samples that were quantified by the TaqMan assay at levels above the expected LLQ of the ExaVir assay. Two samples (2/119; 1.7%) (subtype D and CRF01) showed a VL of 3.1 and 3.9 log₁₀copies/ml, respec-tively, by ExaVir assay but an undetectable VL by TaqMan assay. By RealTime assay, the subtype D sample from a treated patient also showed an undetectable VL, whereas the CRF01 sample showed a VL of 3.2 log₁₀copies/ml. The coefficient of correlation (R) between the two assays was 0.92 overall and was 0.95 and 0.91 for subtype B and non-subtype B samples, respectively (Fig. 1b). The VL measurements differed on av-erage by 0.18 (95% CI, 0.08 to 0.29) log₁₀copies/ml in favor of the TaqMan assay (P⫽0.0005). Pitman’s test of the difference in variance (r) was⫺0.043 (P⫽0.647). In the Bland-Altman comparison, the limits of agreement (the reference range for difference) were ⫺0.94 and 1.31 log₁₀ copies/ml (Fig. 2b). Overall, 5/119 (4.2%) samples (two CRF02, one subtype D,

one CRF01, and one CRF14) fell outside the reference range, including two samples (both CRF02) underquantified by the ExaVir assay and three samples (subtype D, CRF01, and CRF14) underquantified by the TaqMan assay (Table 2).

Comparison of RealTime and TaqMan assays.Overall, 91/

119 (76.5%) samples were quantified by both assays, with a median VL of 4.5 (range, 1.6 to 6.8) log₁₀ copies/ml by RealTime assay and 4.4 (range, 1.8 to 6.8) log₁₀copies/ml by TaqMan assay. There were 25/119 (21.0%) samples with un-detectable VL by both assays, all from treated patients. In addition, one sample (1/120; 0.8%) (subtype A) showed a VL

FIG. 1. Correlation between ExaVir and RealTime assays (a), ExaVir and TaqMan HIV-1 assays (b), and RealTime and TaqMan assays (c), as determined by the parallel testing of 119 samples. All VL values are in log10copies/milliliter. Undetectable VL levels were scored as 40 (1.6

log10) copies/ml, corresponding to the lower limit of the quantification of

the two molecular assays. The linear regression line is shown.

FIG. 2. Bland-Altman analysis of the agreement between ExaVir and RealTime assays (a), ExaVir and TaqMan assays (b), and Real-Time and TaqMan assays (c). The labels show the subtype of samples outside the 95% level of agreement.

TABLE 2. Samples showing HIV-1 plasma VL levels outside the 95% level of agreement between two assays when tested by

ExaVir, RealTime, and TaqMan assays

Subtype

Viral load (log10copies/ml)

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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of 2.2 log₁₀copies/ml by TaqMan assay but an undetectable VL by RealTime assay. Conversely, two samples (2/119; 1.7%) (subtype C and CRF01) showed a VL of 2.3 and 3.2 log₁₀ copies/ml, respectively, by RealTime assay but an undetectable VL by TaqMan assay. The coefficient of correlation (R) was 0.96 overall and was 0.97 and 0.96 for subtype B and non-subtype B samples, respectively (Fig. 1c). The VL measure-ments differed on average by 0.09 (95% CI, 0.02 to 0.17) log10

copies/ml in favor of the RealTime assay (P⫽0.01). Pitman’s test of the difference in variance (r) was 0.194 (P⫽0.035). In the Bland-Altman comparison, the limits of agreement (the reference range for difference) were⫺0.69 and 0.88 log10

cop-ies/ml (Fig. 2c). Overall, 6/119 (5.0%) samples fell outside the reference range, comprising five samples (one subtype B, one subtype D, one CRF01, one CRF02, and one CRF14) under-quantified by TaqMan assay and one sample (subtype D) un-derquantified by RealTime assay (Table 2).

Assay performance with the WHO HIV-1 RNA IS. The

ExaVir assay consistently underquantified the subtype B IS, whereas the TaqMan assay consistently overquantified the IS (Fig. 3). With the ExaVir assay, across seven quantified dilu-tions ranging from 4.4 to 2.6 log10IU/ml, the mean difference

in VL was 0.3 (range, 0.0 to 0.5) log₁₀copies/ml. With the molecular assays, across 10 dilutions ranging from 4.4 to 1.6 log₁₀IU/ml, the average difference was 0.0 (range, 0.1 to⫺0.3) log10copies/ml with the RealTime assay and⫺0.2 (range⫺0.1

to⫺0.3) log₁₀copies/ml with the TaqMan assay.

Reproducibility and specificity of the ExaVir assay.To

as-sess the intraassay reproducibility of the ExaVir assay, 10 sam-ples were tested in duplicate, of which 7 showed a detectable VL. Overall, replicate values differed by a mean of 0.04 (stan-dard error, 0.07) log10copies/ml. Specificity was assessed with

10 HIV-negative plasma samples, all of which showed an un-detectable VL.

DISCUSSION

Molecular VL assays widely used in high-income countries for the virological monitoring of antiretroviral therapy are

dif-ficult to implement in resource-limited settings due to both financial and practical constraints. The ExaVir assay offers a cheaper and simpler methodology for VL measurement in these settings. In this study, the version 3 assay showed an excellent correlation and a high degree of concordance with two widely used commercial real-time PCR assays, a compar-ative performance that was consistent with the relationship that the two molecular assays showed with each other.

Previous studies analyzed the performance of the ExaVir assay versions 1 and 2 and found good detection rates for samples with VL above 10,000 and 400 copies/ml, respectively, and a good overall correlation with molecular assays, most commonly the Roche Amplicor HIV-1 Monitor test version 1.5 (3, 5, 12, 14, 15, 23, 25). An evaluation of the performance of the version 3 assay compared to that of version 2 (and the Roche Amplicor HIV-1 Monitor test version 1.5) was pre-sented in abstract form in 2008 (9). It demonstrated increased sensitivity with version 3 compared to that of version 2, with a mean difference of 0.19 log₁₀ copies/ml. In this study, VL measurements with clinical samples generally were under-quantified by the ExaVir assay compared to quantification by the molecular assays. With the IS, we also observed underquantification by the ExaVir assay, while we detected good performance for the RealTime assay and a small but consistent overquantification by the TaqMan assay. The ExaVir assay quantified 93% of samples with VL of ⬎200 (2.3 log₁₀) copies/ml by both molecular assays and 100% of samples with VL of⬎1,585 (3.2 log10) to 1,995 (3.3 log10)

copies/ml. Thus, performance was in agreement overall with the range of quantification reported by the manufacturers. These findings indicate that the ExaVir assay can reliably identify significant viremia in treated patients.

The significance of low-level viremia continues to be de-bated in high-income countries (16, 18). Interpretation and management likely will be even more challenging in developing countries, where drug options are limited. Previous studies reported stable CD4 cell counts and a low risk of disease progression at VL levels below 4.0 log₁₀ copies/ml (4, 18). However, it would be of importance to consider additional outcome data, including the emergence of drug resistance and the exhaustion of treatment options, in order to establish ap-propriate VL cutoffs that should trigger a treatment change where resources are limited. Meanwhile, an assay with a lower limit of quantification of around 200 copies/ml would be of immediate practical use.

A few samples showed significant differences in VL mea-surements between assays. ExaVir generally underquantified these discrepant samples compared to the quantification of the molecular assays, consistently with the reduced sensitivity of the assay. While a problem with false-positive results was ap-parent in a study of the ExaVir version 2 assay (24), there was no evidence of a significant problem with assay specificity in this study. Among 26 samples with an undetectable VL by the two molecular assays, all from treated patients, only one showed a detectable VL by ExaVir assay, at 1,259 (3.1 log₁₀) copies/ml, while HIV-negative samples all showed an unde-tectable VL by ExaVir assay. One additional sample, from a patient infected with CRF01, was quantified by ExaVir assay as well as by RealTime assay but not by TaqMan assay, suggesting a problem with quantification by the latter. Although

Real-FIG. 3. Comparison of VL measurements obtained by the ExaVir, RealTime, and TaqMan assays with serial dilutions of the WHO sec-ond IS for HIV-1 RNA, ranging from 4.4 to 1.6 log10(25,000 to 40)

IU/ml.

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Time and TaqMan assays showed a high degree of concor-dance, four other samples, comprising subtype B, subtype D, CRF02, and CRF14, were significantly underquantified by TaqMan assay, whereas one subtype D sample was underquanti-fied by RealTime assay. Previous studies suggested an impaired performance of the TaqMan version 1 assay for the quantification of non-subtype B samples (10, 21), and the recently launched version 2 assay promises to address this problem. We previously reported the good overall performance of the RealTime assay (6). In this study, the underperformance of either real-time PCR assay was more common with non-subtype B samples but not consistent with specific subtypes. Taken together, these findings indicate that, despite the significant improvements introduced in recent years, HIV sequence variability continues to challenge molecular VL assays. Testing with a second method is recommended when VL results are not consistent with the patient’s history, and in these circumstances the use of a nonmolecular assay like the ExaVir assay also could be considered.

In summary, we found an excellent correlation and a high degree of concordance between the ExaVir assay and current real-time molecular assays. The increased throughput and re-duced turnaround time, hands-on time, and use of consum-ables with version 3 relative to that of version 2 make the assay an attractive option for the virological monitoring of treated patients where infrastructure is limited. Some of the previously recognized limitations (25) remain, including the large sample volume required for analysis and the lack of automation.

ACKNOWLEDGMENTS

We thank Martyn Eales at Cavidi, Sweden, for assistance with set-ting up the ExaVir assay.

This work was supported by the Royal Free Hampstead NHS Trust Departmental R&D Fund.

W.L. and G.S.C. received travel support from Abbott Diagnos-tics. M.J. received consultancy and speaker honoraria from Abbott Pharmaceuticals and Roche Pharmaceuticals. A.M.G. has received consultancy and speaker honoraria from Abbott Diagnostics, Ab-bott Pharmaceuticals, Roche Molecular Diagnostics, and Roche Pharmaceuticals.

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