Performance attributes of the LCx HCV RNA quantitative assay.

Journal of Virological Methods 115(2004)

207–215

Performance attributes of the LCx ?HCV RNA quantitative assay

Gregor Leckie ?,George Schneider,Klara Abravaya,Robert Hoenle,Julie Johanson,John Lampinen,Reuben Ofsaiof,Lisa Rundle,Shruti Shah,Andrea Frank,Dan Toolsie,

Roy Vijesurier,Hong Wang,John Robinson

Abbott Laboratories,Molecular Diagnostics,D99G AP20,100Abbott Park Road,Abbott Park,IL 60064-6015,USA

Received 9September 2003;accepted 2October 2003

Abstract

The LCx ?HCV RNA quantitative assay (Abbott Laboratories,North Chicago,IL)is designed to use competitive reverse transcriptase-polymerase chain reaction (RT-PCR)and microparticle enzyme immunoassay (MEIA),in combination with a modi?ed Qiagen sample preparation method,to measure the level of hepatitis C virus (HCV)in human plasma and serum.The assay provides quantitative results in international units (IU)of HCV RNA/ml,in copies of HCV RNA/ml,or their log (base 10)equivalents.A conversion study determined that 1IU equals 4.3copies.The LCx HCV assay detected HCV RNA transcripts representative of genotypes 1–6with near equal ef?ciency.The assay did not cross-react with high concentrations of 21potentially cross-reactive microorganisms or with 100HCV-negative specimens.The lower limit of detection was demonstrated to be 23IU/ml.The LCx assay had similar sensitivity to the Roche Amplicor HCV (version 2.0)qualitative assay when used to test panels containing 6,12,23,and 47IU/ml.The assay linear range was shown to extend from 23to 2.3million IU/ml.The intra-assay standard deviation (S.D.)was ≤0.066log IU/ml for the four HCV positive samples tested,while for the same samples the observed inter-assay S.D.was ≤0.075log IU/ml.The overall mean assay quantitation value for seven HCV-positive WHO-standardized Acrometrix NAP linearity panel members was within 0.06log IU/ml of the mean assigned value.The assay was demonstrated to correlate acceptably against the Roche Amplicor HCV monitor test (version 2.0).These data suggest that the assay is standardized appropriately against the WHO standard across its linear range and can be used for quantitation of HCV .In addition,with a sensitivity of 23IU/ml,the assay can be used to determine if post-therapy viral clearance has occurred.?2003Elsevier B.V .All rights reserved.

Keywords:HCV;RT-PCR assay;Viral load

1.Introduction

Hepatitis C virus (HCV),a major etiological agent of hepatitis,is an enveloped virion with a single-stranded,pos-itive sense RNA genome of approximately 9500nucleotides (Choo et al.,1989;Kuo et al.,1989;Cuthbert,1994;Clarke,1997).Up to 85%of HCV-infected individuals develop chronic hepatitis,with approximately 20%of the chronically infected individuals developing cirrhosis (EASL,1999).In patients with cirrhosis,the incidence of hepatocellular carci-noma is 1–4%per year (EASL,1999).HCV infection can be cleared using interferon or peginterferon monotherapy,or us-ing interferon/ribavirin or peginterferon/ribavirin combina-tion therapy (Davis et al.,1998;McHutchinson et al.,1998;

Corresponding author.Tel.:+1-847-937-1547;fax:+1-847-938-8777.

E-mail address:gregor.leckie@https://www.360docs.net/doc/883118077.html, (G.Leckie).

Lindsay et al.,2001;Manns et al.,2001;Fried et al.,2002).HCV anti-therapeutic decisions are made using a wide vari-ety of information,including the clinical presentation of the patient,the results of liver biopsy,the genotype of the in-fecting HCV isolate,and the concentration of HCV present in a patient’s plasma or serum (EASL,1999;NIH,2002).Current recommendations suggest that nucleic acid tests for quantitation or detection of HCV RNA in serum or plasma be used to determine HCV viral load at four differ-ent points–before the initiation of therapy,3–6months after the initiation of therapy,at the termination of therapy,and periodically after therapy-termination (EASL,1999;NIH,2002).Pre-therapy viral load information is used,in com-bination with the genotype of the infecting HCV isolate,to determine the duration of the anti-HCV therapy regimen.Twelve months of interferon/ribavirin combination therapy is recommended if the patient is infected with HCV geno-type 1and has a viral load greater than two million copies

208G.Leckie et al./Journal of Virological Methods115(2004)207–215

of HCV RNA/ml(800,000IU/ml).A6-month course of interferon/ribavirin combination therapy is recommended if the patient either has a genotype2or3infection or has a viral load lower than800,000IU/ml(EASL,1999; Pawlotsky et al.,2000;NIH,2002).Viral load information is used to monitor the response of patients to anti-HCV therapy.A sustained virological response(SVR)is the goal of anti-HCV therapy and is de?ned as virus concentrations less than50IU/ml24weeks after the end of therapy.How-ever,therapy may be discontinued in patients who fail to achieve a two-log(base10)drop in viral load after the?rst 12weeks of therapy(EASL,1999;NIH,2002).

Initial versions of the Roche Amplicor Monitor and Bayer Quantiplex assays,the most widely commercially available quantitative HCV RNA nucleic acid tests,did not produce easily comparable HCV RNA quantitation values (Pawlotsky,1997).Lack of result comparability was caused by unequal quantitation of HCV genotypes1–6,by lack of result–unit standardization,and for the Roche Monitor assay,by underquantitation of specimens containing greater than approximately500,000IU/ml(Pawlotsky,1997).The comparability of HCV RNA quantitation values produced using these two assays has been improved considerably through implementation of assay modi?cations and the de-velopment of the World Health Organization(WHO)First International Standard96/790(Detmer et al.,1996;Mellor et al.,1999;Saldanha et al.,1999;Gretch,2000;Lee et al., 2000;Martinot-Peignoux et al.,2000;Beld et al.,2002; Germer et al.,2002;Ross et al.,2002;Trimoulet et al.,2002). Neither quantitative assay,however,is sensitive enough to determine if viral clearance has occurred and,therefore, both manufacturers have developed sensitive qualititative assays for this purpose(Lee et al.,2000;Ross et al.,2001). The LCx?HCV RNA Quantitative assay uses competitive reverse transcription PCR(RT-PCR)followed by micropar-ticle enzyme immunoassay(MEIA)detection to quantitate HCV in plasma and serum specimens.An internal standard (IS)transcript RNA is introduced into each specimen at the start of sample preparation and is used in the quantitative algorithm.The assay was designed to quantitate HCV RNA in IU/ml,or copies/ml,and also have suf?cient sensitivity to determine if viral clearance has occurred.In this paper, we describe the performance of the LCx HCV RNA quan-titative assay in studies performed at Abbott Laboratories.

2.Materials and methods

2.1.Panels,samples,and microbiological isolates

The genotype1CLB Pelicheck HCV RNA standard was purchased from the Central Laboratory of the Blood (CLB)Transfusion Service(Amsterdam,The Netherlands). The HCV genotype1World Health Organization(WHO) First International Standard96/790was kindly provided by Dr.John Saldanha at the National Institute for Biologi-cal Standards and Control(South Mimms,UK)(Saldanha et al.,1999).The genotype1NAP nucleic acid panel was purchased from Acrometrix(Berkeley,CA);this panel is calibrated against the WHO First International Standard for HCV(Jorgensen and Neuwald,2001).HCV-positive plasma and serum specimens were purchased from ProMedDx LLC (Plainville,MA),Millenium Biotech,Inc.(Fort Lauderdale, FL),and the Sacramento Medical Foundation(Sacramento, CA).HCV-negative plasma and serum samples were pur-chased from ProMedDx LLC.Hepatitis A virus and Es-cherichia coli were obtained from Abbott Laboratories, while Candida albicans(ATCC2091),?avivirus Dengue type4(ATCC VR-1490),rhinovirus16(ATCC VR-283), Pneumocystis carinii(ATCC50381),Staphylococcus aureus (ATCC4012)and Staphylococcus epidermidis(ATCC146) were obtained from the American Tissue Culture Collection. Nucleic acid from these species was puri?ed and quantitated using standard methodologies.Puri?ed nucleic acid repre-senting the following microbiological species was purchased from Advanced Biotechnologies Inc.(Columbia,MD):hu-man adenovirus type5,human cytomegalovirus,Epstein–Barr virus,hepatitis B virus,human herpes virus type6, herpes simplex virus type1,human immunode?ciency virus type1(HIV-1),HIV type2(HIV-2),human T-cell lym-photropic virus type I(HTLV-I),HTLV-II,Mycobacterium avium,vaccinia virus,and varicella-zoster virus.

2.2.Primer and probe sequences and RNA transcripts The PCR target sequence is in the5 untranslated region (UTR)of the HCV genome.The anti-sense reverse primer is 22nucleotides long and is labeled at its5 terminus with the hapten carbazole.The sense forward primer is22nucleotides long and is unlabeled.The sense HCV-speci?c probe is18 nucleotides long and is labeled at its5 end with the hapten adamantane.The sense IS transcript-speci?c probe is18 nucleotides long and is labeled at its5 end with the hapten dansyl.

The HCV RNA transcript and the IS transcript are both 1426bases long.The HCV RNA transcript and IS RNA transcript are identical except for the probe-binding sites. The transcripts were produced as previously described ex-cept that the HCV plasmid and the IS plasmid were lin-earized with Sca I prior to RNA transcription(Johanson et al.,2001).RNA transcripts were quantitated using hyper-chromicity(Collins et al.,1995).

Plasmids containing sequences representing the HCV5 untranslated region(UTR)from genotypes1a,1b,2a,2b,3a, 4,5,and6a were constructed by Dr.Johnson Lau(University of Florida,Gainsville).RNA transcripts were produced using the above methodology.

2.3.Sample preparation

Sample preparation is performed using a modi?ed Qi-agen RNA puri?cation procedure.One ml of specimen

G.Leckie et al./Journal of Virological Methods115(2004)207–215209

(ACD plasma,CPD plasma,EDTA plasma,or serum) or control(Negative Control,Low Positive Control2.37 log IU/ml of HCV RNA transcript,or High Positive Control 5.37log IU/ml of HCV RNA transcript)is added to1ml of guanidine hydrochloride-based lysis buffer that contains approximately3.68log copies of IS RNA transcript.After addition of125?l of protease to each sample(specimen or control),the reactions are incubated for10min at70?C, and then cooled for10min at room temperature.After adding1ml of ethanol,each reaction is added to a Qia-gen spin column that has been placed onto a24-position vacuum manifold.Each sample is passed through the spin column matrix via application of a vacuum(20–28in.of Hg).HCV genomic RNA,HCV transcript RNA,and IS transcript RNA are captured on the spin column matrix. Four vacuum-mediated500?l washes(two with AW1 buffer followed by two with AW2buffer)are performed to remove residual sample contaminants.The spin columns are removed from the vacuum manifold and centrifuged (4min at13,000×g)to remove residual AW2buffer.RNA is eluted from each spin column by addition of120?l of RNAse-free water(Buffer A VE),followed by centrifuga-tion(4min at13,000×g)into an RNAse-free collection tube.

https://www.360docs.net/doc/883118077.html,petitive RT-PCR

Fifty microliters of eluted sample are added to an ampli-?cation vial that contains100?l of pre-aliquoted ampli?ca-tion reagent(100mM bicine pH8.25,16%glycerol,230mM K+,300uM of each deoxynucleoside triphosphate(dATP, dCTP,dGTP,dTTP),30–70nM HCV speci?c unlabeled forward primer,125nM HCV speci?c carbazole-labeled reverse primer,100nM HCV-speci?c adamantane-labeled probe,100nM internal standard-speci?c dansyl-labeled probe,0.02mg/ml acetylated BSA,0.125%Tween20, 0.04%sodium azide and10.5units recombinant Thermus thermophilus DNA polymerase).Thus,each?nal reaction contains approximately 3.31log copies of IS transcript RNA,plus HCV RNA that was present in the sample or controls.50?l of activation reagent(10mM manganese chloride,0.045%sodium azide,0.01%xylenol orange dye) is added to each ampli?cation vial and activated reactions are maintained in a chilled rack prior to being placed into a Thermal Cycler(Perkin Elmer480).Competitive RT-PCR is performed using the following thermal cycling program:

(1)Reverse transcription(1cycle:94?C for1min,62?C for

30min,94?C for2min)in which the carbazole-labeled reverse primer initiates reverse transcription from both the IS transcript RNA,as well as HCV genomic RNA, if present.

(2)Lower stringency PCR(5cycles:94?C for15s,

58?C for20s with automatic extension of1s per cycle).(3)Higher stringency PCR(40cycles:94?C for15s,62?C

for25s with automatic extension of1s per cycle). (4)Oligonucleotide hybridization(1cycle:97?C for

5min,15?C for5min)in which the HCV-speci?c probe and the IS-speci?c probe bind to their respective amplicons.

(5)Storage prior to detection(15?C soak).

2.5.Detection

Following RT-PCR,the ampli?cation vials are placed into an LCx?Analyzer,a24-position automated batch analyzer.In the LCx Analyzer,an aliquot of each sample is transferred to a reaction cell,where HCV and IS am-pli?cation products are captured by anti-carbazole-coated microparticles via the carbazole-labeled reverse primer. The microparticle complexes are transferred to a glass ?ber matrix to which the microparticle complexes bind irreversibly.After washing,the bound microparticle com-plexes are incubated with a mix of anti-adamantane alkaline phosphatase conjugate and anti-dansyl beta-galactosidase conjugate.The anti-adamantane alkaline phosphatase con-jugate binds only to the HCV-speci?c probe while the anti-dansyl beta-galactosidase binds only to the IS probe. The detection of IS ampli?cation product is accomplished through cleavage of the substrate,7-beta galactopyra-nosyl coumarin-4-acetic acid-(2-hyroxyethylamide)(AUG) by beta-galactosidase to produce a?uorescent product, 7-hydroxy-4-[acetic acid-2-hydroxyethylamide]coumarin (AU),that is measured by the LCx Analyzer optical as-sembly.After measuring the signal generated by AU,the LCx Analyzer washes the glass?ber matrix and adds 4-methylumbelliferyl phosphate(MUP)for the detection of HCV ampli?cation product.The MUP is cleaved by alkaline phosphatase to4-methyl umbelliferone(MU). The LCx Analyzer optical assembly measures the re-sultant?uorescence.At the completion of detection,the LCx Analyzer delivers two reagents(a chelating metal complex and an oxidizing reagent)into the LCx reaction cells.Addition of these two reagents,results in a mil-lionfold reduction of the nucleic acids present(Hu et al., 1996).

2.6.Calibration and result calculation

Two types of runs are performed using the LCx Analy-zer—calibration and mode2.Both types of runs contain one replicate each of negative control,low positive control, and high positive control.Calibration runs also contain two replicates each of six different calibrators.Unlike controls, calibrators are not processed through sample preparation. Rather,50?l of each calibrator,containing2000copies (log3.31copies/ml)of IS transcript RNA and various con-centrations of HCV transcript RNA(equivalent to0,1.67, 2.67,3.67,4.67,and6.67log IU/ml),are added to an am-pli?cation vial,followed by addition of50?l of activation

210G.Leckie et al./Journal of Virological Methods 115(2004)

207–215

LCx HCV RNA Quantitative Assay Example Calibration Curve

0500

1000

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A v e r a g e L C x R a t e (c /s /s )

LCx HCV RNA Quantitative Assay Example Calibration Curve

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Calibrator Concentration (log IU/ml)

L o g R a t i o

(a)

(b)

Fig.1.(a)Example of rates used to generate an LCx HCV RNA quantitative assay calibration curve,demonstrating the competitive relationship between

internal standard rates (AUG signal)and HCV rates (MUP signal).(b)Example of a calibration curve generated by taking the log (HCV rate/IS rate)from the data in Fig.1a .

reagent.The calibrators are matched to the CLB Pelicheck HCV RNA standard.After ampli?cation and detection,the log rate ratio (LRR)of each calibrator replicate is calculated (log 10(HCV rate/IS rate)).Because the RT-PCR reaction is competitive,samples with low HCV concentrations produce high AUG signals and low MUP signals,while samples with high HCV concentrations produce low AUG signals and high MUP signals (Fig.1a ).The mean LRR of each calibrator level is stored within the LCx Analyzer software (Fig.1b ).Specimens can be run on Calibration runs (max-imum of nine specimens)or on mode two runs (maximum of 21specimens).To determine their concentration,the LRR of each specimen is calculated and compared against the LRR values of the stored calibration curve.Because the concentrations of the calibrators are known,the specimen LRR values can be converted into concentration https://www.360docs.net/doc/883118077.html,e of conversion factors permits HCV RNA concentration results to be reported in copies/ml or IU/ml,or their log-arithmic equivalents.It is important to note that the LCx Analyzer only reports specimen results when the run con-trols,and the internal control for the sample in question,are valid.

G.Leckie et al./Journal of Virological Methods 115(2004)207–215211

3.Results

3.1.Standardization and lower limit of detection (LLD)Seven replicates each of the CLB genotype 1Pelicheck HCV-RNA standard and the WHO HCV genotype First In-ternational Standard were quantitated on the same assay run,and a conversion factor to compare the two standards was determined to be one WHO IU equals

4.3CLB copies (95%con?dence interval of 3.7–

5.1).The lower limit of detection (LLD)of the assay was assessed using three reagent lots and three instrument pairs (LCx Thermal Cycler and LCx Analyzer).Each reagent lot and instrument pair was used to test 10replicates of panel members containing 4

6.5IU/ml and 23.3IU/ml,15replicates of panel members containing 11.6IU/ml and 5.8IU/ml,and 35HCV-negative specimens.The HCV positive panel members were created by dilution of the CLB standard.The upper limit of the negative popula-tion (ULN)was de?ned as the LRR value that encompasses 99%of the assay values resulting from the testing of the HCV-negative specimens.The assay lower limit of detection (LLD)was calculated for each reagent lot and instrument pair,and was de?ned as the concentration of HCV where 95%of the results were above the ULN.The reagent lot and instrument pair LLD values were 5.8,10.7,and 6.0IU/ml.To ensure that the LLD could be maintained with all reagent lots,the assay software reports quantitative results if the HCV concentration is greater than or equal to 23IU/ml,or 1.37log IU/ml.Because quantitation values below 23IU/ml are not reported by the assay software,a specimen contain-ing exactly 23IU/ml would be quantitated 50%of the time.The LCx HCV RNA Quantitative assay sensitivity was compared to that of the Roche Amplicor HCV (version

2.0)

Linear Range

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O b s e v e d C o n c e n t r a t i o n (l o g I U /m l )

Fig.2.Correlation between observed concentrations and actual concentrations of HCV-positive samples,as measured by the LCx HCV RNA quantitative assay.

Table 1

Comparative sensitivity of the LCx HCV RNA quantitative assay and the Roche Amplicor HCV qualitative test when used to test the same panels Panel

concentration (IU/ml)Percentage of positive (≥LLD)Abbott LCx HCV results (%)Percentage of positive Roche Amplicor HCV qualitative test results (%)47100(12/12)66.7(8/12)2358.3(7/12)75(9/12)120(0/12)8.3(1/12)6

0(0/12)

qualitative test that has a claimed sensitivity of 50IU/ml.The CLB standard was diluted in negative plasma to 47,23,12,and 6IU/ml (assuming that 1IU equals 4.3copies).The LCx assay was used to quantitate 12replicates of each of the panels at Abbott Laboratories,and each panel member was quantitated 12times using the Roche Amplicor test at a clin-ical laboratory.The data are summarized in Table 1.These data show that the LCx assay quantitated 100%(12/12)of the replicates at 47IU/ml and 58.3%(7/12)of the replicates at 23IU/ml.The Roche Amplicor HCV qualitative test de-tected 66.6%(8/12)of the replicates at 47IU/ml and 75%(9/12)of the replicates at 23IU/ml.3.2.Linear range and ability to quantitate a therapy panel

The linear range of the assay was assessed using three dif-ferent reagent lot-instrument pairs.Each reagent lot-instru-ment pair was used to test ten replicates each of panel members containing the following HCV concentrations:1.07,1.37,1.67,2.37,3.37,4.37,5.37,6.37,6.67,and 6.97log IU/ml.Illustrated in Fig.2are representative linearity

212G.Leckie et al./Journal of Virological Methods115(2004)207–215 Table2

Intra-assay and inter-assay precision of the Abbott LCx HCV RNA quan-

titative assay

Panel member N Mean

concentration

(log IU/ml)

Intra-assay S.D.

(log IU/ml)

Inter-assay S.D.

(log IU/ml)

150 1.920.0660.075

250 2.620.0310.036

350 3.330.0550.070

450 5.710.0510.057

data for one reagent lot and instrument pair.Fig.2illustrates that the quantitative assay response?attens above 6.37 log IU/ml and quantitative assay results are therefore re-ported from the LLD to6.37log IU/ml.If a sample contains greater than6.37log IU/ml,the sample can be diluted1:50 and retested to provide a result within the linear range of the assay,thus extending the linear range to8.07log IU/ml.

3.3.Precision

Assay precision was assessed with two sets of reagent lot and instrument pairs.Each reagent lot and instrument pair was used to test?ve replicates each of a four-member precision panel on5different days.The data are presented in Table2.For the combined reagent lot and instrument pair data,the intra-assay standard deviation(S.D.)for each panel member was≤0.066log IU/ml,while the inter-assay S.D.s (includes intra-assay S.D.)were≤0.075log IU/ml.

3.4.Con?rmation of standardization and

analytical performance

To verify the standardization,LLD,and linear range,the genotype1NAP nucleic acid panel from Acrometrix was tested using one reagent masterlot-instrument pair.Each of the seven HCV-positive panel members(1.70,2.70,3.70, 4.70,5.30,5.70,and6.30log IU/ml)was tested eight times. The LCx assay quantitated all eight replicates of each panel member,including the panel member at1.7log IU/ml.In Table3,it can be seen that quantitation differences be-tween the expected panel member log IU/ml values and the mean observed log IU/ml values ranged from?0.18 Table3

Con?rmation of standardization and analytical performance

Panel ID Assigned value

(log IU/ml)

Observed value

(log IU/ml)

Mean difference

(assigned?observed)

PM1 1.70 1.490.21

PM2 2.70 2.78?0.08

PM3 3.70 3.680.02

PM4 4.70 4.79?0.09

PM5 5.30 5.48?0.18

PM6 5.70 5.82?0.12

PM7 6.30 6.46?0.16

Overall mean difference=?0.06.Line equation:y=1.0675x?0.2331, r=0.999.to0.21log IU/ml.The overall mean difference was?0.06 log IU/ml and the correlation coef?cient(r)was0.999.These data support that the assay is standardized appropriately against the WHO standard and can meet the claimed LLD and ULQ.

3.5.Cross-reactivity and speci?city

To assess potential assay cross-reactivity,6.00log copies/ ml of each microorganism,or its equivalent in puri?ed genomic nucleic acid,was processed through sample preparation prior to performing one replicate of assay test-ing.No cross-reactivity was observed when the following microorganisms were tested:human adenovirus type5, Candida albicans,human cytomegalovirus,Epstein–Barr virus,Escherichia coli,the?avivirus,Dengue type4, hepatitis A virus,hepatitis B virus,human herpes virus type6,herpes simplex virus type1,human immunode?-ciency virus type1(HIV-1),HIV type2(HIV-2),Human T-Cell Lymphotropic Virus Type I(HTLV-I),HTLV-II, Mycobacterium avium,Pneumocystis carinii,rhinovirus 16,S taphylococcus aureus,Staphylococcus epidermidis, vaccinia virus,and varicella-zoster virus.In addition,100 anti-HCV antibody-negative specimens were negative when tested by the assay.

3.6.Genotype detection

To assess whether the assay detects HCV genotypes with equal ef?ciency,puri?ed RNA transcripts represen-tative of HCV genotypes1a,1b,2a,2b,3,4,5,and6 were diluted to2.37log IU/ml and tested in the assay. Each genotype transcript was added directly to six different ampli?cation vials before ampli?cation and detection.The data presented in Fig.3show that the reported assay mean log IU/ml values were2.38,2.39,2.39,2.43,2.42,2.42, 2.49,and2.57for genotypes1a,1b,2a,2b,3,4,5,and6,

respectively.

Genotype Detection

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)

Fig.3.Detection of HCV genotypes1a,1b,2a,2b,3,4,5,and6by the LCx HCV RNA quantitative assay.Error bars represent the standard deviation of the observed concentrations of each genotype.

G.Leckie et al./Journal of Virological Methods 115(2004)207–215

213

Correlation vs. Roche Monitor

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A b b o t t L C x (l o g I U /m l )

Fig.4.Correlation of the Abbott LCx HCV RNA quantitative assay with Roche Amplicor HCV monitor test,version 2.0.

3.7.Correlation with available commercially quantitative assays

Assay correlation was determined by testing purchased HCV-positive specimens that had been quantitated using the Roche Monitor v.2.0test (n =42).These specimens had also been genotyped (?ve 1a,?ve 1b,one 2a/2c,four 2b,two 3a,one 3e,174,one 4a,one 4b/4c,one 4c/4d,two 5a,and two 6a).Comparing the LCx HCV log IU/ml quan-titation values and the Roche log IU/ml values (Fig.4),the correlation coef?cient,r ,was 0.9and the line equation was y =0.8439x +0.5458.All but one of the LCx and Roche quantitation log IU/ml values were within one log of each other.One genotype 2b sample was quantitated 1.5log IU/ml lower by the LCx assay than by the Roche assay.This spec-imen was quantitated using the Bayer version 3.0assay and it produced a quantitation value within 0.1log IU/ml of the LCx assay value.The mean LCx HCV log IU/ml value was 0.32log lower than the mean Roche quantitation value.

Table 4

Change in serum HCV RNA for a patient undergoing interferon therapy as measured by the LCx HCV RNA quantitative assay and the Bayer Quantiplex version 2.0assay Date of draw bDNA v.2.0log (copies/ml results)LCx log

(copies/ml results)4/5/997.447.684/7/997.557.434/9/99 6.92 6.724/12/99<5.30 4.374/14/99<5.30 3.364/16/99

<5.30

3.81

In addition,the LCx HCV assay was used to quantitate six members of a panel that was collected serially from a HCV positive patient prior to,and after,initiation of interferon therapy on 4/11/99.Table 4shows the LCx assay data as compared to the log copies/ml values produced by the Bayer Quantiplex version 2.0.The post-interferon therapy panel members were below the 5.30log copies/ml sensitivity limit of the bDNA v.2.0assay.

4.Discussion

The LCx HCV RNA Quantitative assay was designed to produce accurate and reproducible quantitation results for HCV genotypes 1–6.The assay primers and probes were designed to hybridize to sequences within the 5 untranslated region of HCV ,the most highly conserved region of the HCV genome.To help achieve reproducible genotype quantitation,the ?rst ?ve PCR thermal cycles are carried out at a lower stringency than the subsequent PCR cycles.The LCx HCV assay quantitated genotypes 1–6HCV RNA transcripts equally,and produced similar quantitation values as the Roche Monitor v.2.0test when used to assay 42patient specimens containing represen-tatives of genotypes 1–6.This is signi?cant because the Roche Amplicor HCV Monitor test (version 2.0)test has been demonstrated to quantitate HCV genotypes 1–6with equality (Mellor et al.,1999;Lee et al.,2000).The LCx HCV RNA quantitative assay did not cross-react with high concentrations of microorganisms that might be present in a plasma or serum specimen,and produced a speci?city of 100%when used to test HCV negative plasma samples.In combination,these data suggest that the LCx HCV assay

214G.Leckie et al./Journal of Virological Methods115(2004)207–215

quantitates HCV genotypes1–6equally with excellent speci?city.

The LCx HCV assay is linear from the LLD of1.37 log IU/ml to the ULQ of6.37log IU/ml.The relatively wide linear range is achieved by using the ratio of the HCV sig-nal divided by the internal standard signal.The assay lin-ear range encompasses the5.9log IU/ml(800,000IU/ml) threshold value for therapy(Pawlotsky et al.,2000).Some specimens will contain a concentration of HCV greater than the ULQ of the LCx HCV RNA Quantitative assay,but these specimens can be diluted1:50and retested.

The intra-assay S.D.of the LCx HCV RNA quantita-tive assay was≤0.066log IU ml for all four HCV posi-tive samples tested,which is in line with the intra-assay S.D.of≤0.15log copies/ml required to differentiate half-log changes in viral load(Yen-Lieberman et al.,1996).Unlike monitoring of HIV anti-viral therapy,where the ability to detect a half log shift in viral load is important,a two-log decrease in viral load is considered clinically signi?cant in HCV anti-viral therapy monitoring(NIH,2002).

The LCx HCV assay calibrators were standardized against the CLB genotype1Pelicheck HCV RNA copies per ml standard and an assay conversion factor between the CLB copies per ml and the WHO IU per ml was established to be 4.3copies to1IU.The95%con?dence limits of this con-version factor of3.7–5.1,encompass the conversion factor of3.8copies to1IU reported by the developers of the CLB and WHO standards(Saldanha et al.,2000).Further con?r-mation of the accuracy of this LCx HCV conversion factor was obtained by running the WHO-standardized Acrometrix linearity panel(Jorgensen and Neuwald,2001).The over-all mean LCx HCV RNA quantitative assay result differed from the overall expected value by?0.06log IU/ml.

The correlation data between the LCx HCV RNA quan-titative assay and the Roche Amplicor HCV monitor test (version2.0)are limited.However,the high observed corre-lation coef?cient of0.90(LCx–Roche)suggest that both as-says perform well to quantitate genotypes1–6with equality, and produce precise results over their claimed linear ranges. The mean Roche Amplicor HCV monitor test(version2.0) log IU/ml value was0.32log(or approximately two-fold) higher than that produced by the LCx HCV RNA quantita-tive assay,which could be explained by use of,in this study, archived instead of newly generated Roche quantitation val-ues,by lot-to-lot variability,or by differences in standard-ization strategy.One report suggested the Roche Amplicor HCV monitor test(version2.0)over-quantitated the WHO standard by0.47–0.49log IU/ml(Pisani et al.,2002).

The Acrometrix panel testing data support that the LCx HCV RNA quantitative assay has a LLD of1.37log IU/ml (23IU/ml),and thus could be used to determine if HCV viral clearance had occurred.HCV is considered cleared if virus levels fall below50IU/ml of HCV RNA/ml(NIH, 2002).Additionally,the sensitivity of the LCx HCV RNA quantitative assay was compared to that of the Roche HCV Amplicor qualitative assay and had similar sensitivity for samples containing47IU/ml.These data suggest that the LCx HCV RNA quantitative may be used both to quantitate HCV in plasma and serum,and to determine if anti-HCV therapy has resulted in clearance of HCV.

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