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Contents lists available at ScienceDirect

Journal of Clinical Virology

j ourna l h om epage: www.elsev ier .com/ locate / j cv

etection limit of architect hepatitis C core antigen assay inorrelation with HCV RNA, and renewed confirmation algorithm foreactive anti-HCV samples

ornelia Ottiger ∗, Nicole Gygli, Andreas R. Huberantonal Hospital of Aarau, Department of Laboratory Medicine, CH-5001 Aarau, Switzerland

r t i c l e i n f o

rticle history:eceived 29 June 2013eceived in revised form 7 August 2013ccepted 26 August 2013

eywords:epatitis CCV core antigenCV RNACV viral loadCV PCR

a b s t r a c t

Background: hepatitis C infections are detected by anti-HCV screening tests. Reactive anti-HCV resultsgive no information about the presence or absence of hepatitis C viruses, or of unspecific reactivity. Toobtain information about the viral load, HCV RNA measurements, following a reactive anti-HCV result,are performed in well equipped and specialised laboratories. Anti-HCV immunoblots are the only meansto exclude non specific reactivity. The measurement of HCV core antigen (HCV-Ag), as an alternative toHCV RNA, is discussed, as it can be analysed on the same instrument as anti-HCV.Objectives: The detection limit of HCV-Ag is crucial to use it in lieu of HCV RNA, in regard of the differentgenotypes. A renewed algorithm is proposed to exclude unspecific reactivity of anti-HCV.Study designs: Samples were tested on Architect i2000SR (Abbott) for anti-HCV and HCV-Ag. HCV RNAmeasurements were obtained by Cobas Ampliprep/Taqman (Roche) or m2000rt® (Abbott).

nti-HCVCV genotyperchitect

Results and Conclusions: Comparison between HCV-Ag and HCV RNA from 126 samples of 101 patientswith chronic hepatitis C gave linear regression R2 0.89, slope 0.885 and intercept −2.258, which wereindependent of the genotypes. The detection limit of HCV-Ag was between 2.4 and 4.5 Log10 IU/mL. Arenewed algorithm for confirmation of reactive anti-HCV results is proposed: active or resolved hepatitisC infections or false reactivity can be differentiated by sequenced reflex testing due to HCV-Ag, anti-HCVimmunoblot and HCV RNA.

. Background

Hepatitis C virus (HCV) infection is usually diagnosed by theetection of anti-HCV antibodies. The window period between

nfection and seroconversion remains approximately 70 days [1].or earlier diagnosis of infection, nucleic acid technology-basedests (PCR) have been developed to reduce the window period,nd/or to confirm an active hepatitis C when HCV RNA is detectable2]. Apart from the window period, where a hepatitis C infection cane missed by anti-HCV screening tests, a reactive anti-HCV result

ndicate a resolved, in the absence, or an acute or chronic hepatitis Cn the presence of viruses, respectively. The specificities of the anti-

Please cite this article in press as: Ottiger C, et al. Detection limit of architrenewed confirmation algorithm for reactive anti-HCV samples. J Clin Viro

CV tests are >99%, in which a confirmatory test seems no longere required [3]. However, some patients will be falsely classifiedaving had a hepatitis C infection, if a confirmatory test does not

Abbreviations: HCV Ag, hepatitis C virus core antigen; HCV RNA, hepatitis C virusibonucleic acid; CV%, coefficient of variation; EDTA, Ethylene diaminetetraaceticcid.∗ Corresponding author. Tel.: +41 062 838 52 63; fax: +41 062 838 52 62.

E-mail address: cornelia.ottiger@ksa.ch (C. Ottiger).

386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jcv.2013.08.028

© 2013 Elsevier B.V. All rights reserved.

follow the initial screening test. A confirmation of positive anti-HCVantibodies by anti-HCV immunoblots [4] would be useful where noHCV RNA is detected. Pregnant women and patients with nephro-logical or rheumatoid diseases give often false positive anti-HCVscreening tests. Our observations hinted that a remarkable amountof reactive anti-HCV samples were false positive. Therefore, we sys-tematically tested all reactive anti-HCV samples with confirmatorytests, and we propose an algorithm for a stepwise approach.

The measurement of HCV RNA is used, first, in differentiatingbetween a resolved and an active hepatitis C, and second, for thefollow up under antiviral treatment [5,6]. However, sensitivity toHCV therapy is variable, because of genetic diversity of the viruses,i.e. genotypes, but also of the host, as polymorphisms in the IL28Bwere found to influence the elimination of the viruses [7–9].

Antigenic characterisation showed the potential of HCV coreantigen (HCV-Ag) as a diagnostic marker [10]. HCV-Ag tests weredeveloped as a cheaper alternative in lieu of HCV PCR for confirma-tion, as well as follow-up under antiviral therapy. These assays are

ect hepatitis C core antigen assay in correlation with HCV RNA, andl (2013), http://dx.doi.org/10.1016/j.jcv.2013.08.028

based on the principle of immunoassays, such as ELISA or chemi-luminescence, and are performed with the same techniques andinstruments as anti-HCV. It has been shown that HCV infection canbe diagnosed earlier using the HCV-Ag assay reaching a reduction

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ARTICLECV-2842; No. of Pages 6

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n the window period of about 35 days comparable to that obtainedith amplification techniques [11,12].

Several studies have been conducted to compare HCV RNA andCV-Ag measurements in detecting an active hepatitis C earlier

han with anti-HCV antibodies alone. The first generation of HCV-g became positive when HCV RNA was >104 IU/mL [13,14]. Newerssays, such as Abbott Architect based on chemiluminescence, haveecome available since then. It urges to compare the newer HCV-Agest versus HCV RNA measurements.

. Objectives

Our aim was to correlate HCV-Ag with HCV RNA. The approachas similar to other [15], but with the focus on the detection limit of

he HCV-Ag and the correlation with each genotype. Additionally,e propose a renewed algorithm for the confirmation of anti-HCVith HCV-Ag and/or HCV RNA and anti-HCV immunoblot.

. Study design

First, samples from patients, who were tested for HCV RNA forollow up of their chronic hepatitis C infection, were prospectivelytored at −20 ◦C. Those 126 samples with detectable HCV RNA wereeasured batch wise in triplicates for HCV-Ag. 5 additional sam-

les were used for a diluting experiment, each in one batch. Theenotypes were analysed on that same sample or had been donen a previous one.

Second, 13,099 consecutive samples from routinely orderednti-HCV antibody screening were performed on the automatedlatform Architect i2000SR® (Abbott, Baar, Switzerland). The assay

s based on chemiluminescence technology, where two recom-inant HCV antigens are used for the detection of the anti-HCVntibodies. S/CO < 1 means “not reactive”, and >1 “reactive”, where

is the signal of the sample and CO of the cut-off control.Those 221 samples with first time detected “reactive” anti-

CV (from 13,099 samples), were followed by HCV-Ag, anti-HCVmmunoblot and/or HCV RNA.

HCV-Ag levels were measured on the same Architect i2000SR.he assay consists of monoclonal anti-HCV particles which bind tohe HCV-Ag. Signal intensities are calculated against a calibrationurve, where values <3 fmol/L (<0.477 Log10 fmol/L) are considerednot reactive”.

For HCV RNA measurements, samples were performed either onobas AmpliPrep/Cobas TaqMan® (Roche, Rotkreuz, Switzerland)AP/CTM version 1 for samples of genotype 1, 2 and 3, and ver-ion 2.0 for genotype 4; or extraction was done on BiomerieuxasyMag® with amplification and quantification on m2000rt®

Abbott), respectively.Genotypes were analysed by VERSANT® HCV Genotype 2.0

ssay (LiPA) (Siemens, Zurich, Switzerland). recomLine HCVmmunoblots (Mikrogen, Neuried, Germany) were performed asescribed by the manufacturer.

Statistical analyses were calculated with GraphPad Prism®

oftware version 6 (GraphPad, San Diego, USA). Weighted linearegression analyses were used for statistical analysis of the datand a P value of <0.05 was considered to be significant.

. Results

One focus of this study was to determine the detection limit ofhe HCV-Ag versus HCV RNA. To attain this object, first, HCV RNA

Please cite this article in press as: Ottiger C, et al. Detection limit of architrenewed confirmation algorithm for reactive anti-HCV samples. J Clin Viro

rom 126 EDTA-samples from 101 patients with known chronicepatitis C infections were obtained either on Abbott m2000rt® (72amples) with a RNA detection limit of 1.0 Log10 IU/mL (10 IU/mL),r on Cobas AmpliPrep/TaqMan® Roche system with version 1.0 (54

PRESSVirology xxx (2013) xxx– xxx

samples) with detection limit 15 IU/mL, i.e. 1.2 Log10 IU/mL. Those8 samples of genotype 4, which had been measured with Rocheversion 1.0 before, were repeated when version 2.0 became avail-able, as underestimation of genotype 4 with version 1.0 has beenshown [16]. The samples were compared with HCV-Ag on Architecti2000SR®.

The samples contained RNA between 1.0 and 7.41 (Log10 IU/mL),and were of genotypes 1A, 1B, 2 (non distinguishable subtypes A/C,and B), 3A and 4 (non distinguishable subtypes A/C/D). The corre-sponding HCV-Ag gave values between −2.0 and 4.3 (Log10 fmol/L).

Linear regression from all samples together gave R2 of 0.887with slope 0.892 ± 0.017 and intercept −2.292 ± 0.095 (Fig. 1a).Regression data of each singular genotype were very similar tothe overall data (Fig. 1b–f), and were statistically not signifi-cantly different (P > 0.05). From the pooled slopes and intercepts(Tables 1a and b), HCV RNA can thus be extrapolated by measuringHCV-Ag:

HCVRNA(log10IU/mL) =(HCVAg(log10fmol/L) + 2.258)

0.885(all samples),

or(HCVAg(log10fmol/L) + 2.556

0.935(one sample per patient),

respectively. (1)

From this calculation 6 samples were excluded (3 from the samepatient) as they had been identified as outliers. They had showntoo low HCV-Ag values in comparison with high HCV RNA (Fig. 1e).Whether this finding has a systematic aspect due to a particulargeno-subtype (patients were not under antiviral therapy), or anerror in measuring HCV-Ag or HCV RNA, respectively, is unclear.

To avoid a bias due to several (maximal 3) samples from thesame patient, data were recalculated taking the first sample fromeach patient (Table 1b).

At the point where HCV-Ag becomes negative, i.e.0.477 Log10 fmol/L (3 fmol/L), HCV RNA was still detectable inall samples (Fig. 1 and Tables 2a and b). Slight differences wereseen among the genotypes, which seemed due to the smaller dataset of genotype 2, and of genotype 3A with its generally greatervariance.

HCV-Ag values below the detection limit were found in 21 sam-ples of 14 patients (of total 126 samples), where 9 patients wereunder antiviral treatment.

Five other EDTA-samples, each with a different genotype and ahigh HCV viral load, were chosen for a diluting experiment in deter-mining the detection limit of HCV-Ag. The undiluted samples hadHCV RNA between 5.36 and 6.95 Log10 IU/mL and HCV-Ag between2.08 and 3.86 Log10 fmol/L, and thus were then stepwise diluted inanti-HCV- and HCV RNA-negative blood donor plasma to as low as2.4–2.7 Log10 IU/mL HCV RNA, which gave then results of HCV-Agbelow the cut-off.

The 5 samples, with genotypes 1A, 1B, 2, 3A and 4,at the detection limit of the HCV-Ag, gave HCV RNA ofstill 3.54 ± 0.09 Log10 IU/mL, i.e. 3467 IU/mL (min. 2183, max.5508 IU/L). Values were linearly related with slopes of 1.05 ± 0.05,intercept of −3.27 ± 0.26, and correlation R2 was 0.987 ± 0.01(Fig. 2).

The coefficient of variation (CV%) of the Abbott HCV-Ag assaygave CV of less than 10% above the cut-off of 0.47 Log10 fmol/L. TheCV was higher below the cut-off, which is not unexpected due togreater variances near zero fmol/L (Fig. 3).

ect hepatitis C core antigen assay in correlation with HCV RNA, andl (2013), http://dx.doi.org/10.1016/j.jcv.2013.08.028

Second, the utility of HCV-Ag, following a reactive anti-HCV,as confirmatory test was addressed. Therefore, 13,099 consecutiveroutine samples were screened for anti-HCV. Anti-HCV had alreadybeen formerly confirmed being positive in 191 cases, and had been

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F Abbotl ) � tof

taHp

ig. 1. Comparison of HCV core antigen (HCV-Ag) by chemiluminescence assay on

inear regression model (GraphPad Prism®) for each genotype (a) all genotypes (GTrom calculation, but shown in the graphics (. . .).

Please cite this article in press as: Ottiger C, et al. Detection limit of architrenewed confirmation algorithm for reactive anti-HCV samples. J Clin Viro

hus unnecessarily re-screened. 221 samples with unknown hep-titis C status with “reactive” anti-HCV results were analysed forCV-Ag. The presence of HCV viruses was proofed with HCV-Agositivity in 117 (53%) cases, without the necessity to perform

t Architect® versus HCV RNA. Statistical analysis was performed with least squaresgether; (b) 1A (�); c: 1B (©); d: 2 (�); e: 3A (�;) f: 4 (�). 6 samples were excluded

ect hepatitis C core antigen assay in correlation with HCV RNA, andl (2013), http://dx.doi.org/10.1016/j.jcv.2013.08.028

molecular testing. A minimal viral load or unspecific reactivitycould not be excluded in the remaining 104 (47%) samples with“reactive” anti-HCV and negative HCV-Ag. They needed furtherconfirmatory testing with HCV RNA measurements and anti-HCV

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Table 1aThe regression analysis between HCV RNA (x) and HCV-Ag (y) for each genotype was performed by linear regression from 120 samples of 97 patients.

Number (N) Slope Intercept Correlation R2

Genotype 1A 38 0.873 (±0.027) −2.173 (±0.153) 0.901Genotype 1B 25 0.931 (±0.038) −2.538 (±0.206) 0.891Genotype 2 (A/C, B) 12 0.907 (±0.079) −2.365 (±0.474) 0.804Genotype 3A 30 0.890 (±0.036) −2.332 (±0.189) 0.879

(36)** (0.770 (±0.054)) (−2.056 (±0.294)) (0.662)Genotype 4 (A/C/D) 15 0.820 (±0.033) −1.750 (±0.203) 0.935All genotypes pooled* 0.885 −2.258

* Correlation data not significant (P > 0.05).** Additional 6 samples from 4 patients of genotype 3A (outliers).

Table 1bThe regression analysis between HCV RNA (x) and HCV-Ag (y) for each genotype was performed by linear regression from 97 patients, each with his first sample.

Number (N) Slope Intercept Correlation R2

Genotype 1A 30 0.959 (± 0.033) −2.669 (±0.185) 0.907Genotype 1B 19 1.000 (± 0.052) −2.936 (±0.282) 0.871Genotype 2 (A/C, B) 10 0.904 (± 0.085) −2.362 (±0.501) 0.802Genotype 3A 23 0.977 (± 0.057) −2.882 (±0.327) 0.821

(27)** (0.821 (± 0.085)) (−2.288 (±0.498)) (0.550)Genotype 4 (A/C/D) 15 0.820 (± 0.033) −1.750 (±0.203) 0.935All genotypes pooled* 0.935

* No significance between the genotypes (P > 0.05), pooled slopes and intercepts.** Additional 4 patients of genotype 3A (outliers).

Table 2aHCV RNA at the cut-off value of HCV-Ag of 0.477 Log10 fmol/L, i.e. 3 fmol/L, extrap-olated from 120 samples, within 95% confidence interval (95%CI).

HCV RNA

Log10 (IU/mL) IU/mL

Genotype 1A 2.984 − 3.545 963 − 3507Genotype 1B 3.184 − 3.805 1527 − 6382Genotype 2 (A/C, B) 2.755 − 4.548 568 − 35′318Genotype 3A** 3.037 − 3.692 1088 − 4920Genotype 4 (A/C/D) 2.400 − 3.458 251 − 2870All genotypes pooled* 3.359 2288

* Correlation data not significant (P > 0.05).** Six samples from 4 patients of genotype 3A were excluded from the calculation.

Table 2bHCV RNA at the cut-off value of HCV-Ag of 0.477 Log10 fmol/L, i.e. 3 fmol/L, extrap-olated from 97 patients, within 95% confidence interval (95%CI).

HCV RNA

Log10 (IU/mL) IU/mL

Genotype 1A 3.190 − 3.766 1548 − 5834Genotype 1B 3.352 − 4.073 2249 − 11,830Genotype 2 (A/C, B) 2.627 − 4.648 423 − 44,463Genotype 3A 3.364 − 4.275 2312 − 18,836Genotype 4 (A/C/D) ** 2.400 − 3.458 251 − 2870

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HCV RNA, with no significant differences (P > 0.5) of slopes andintercepts among the genotypes 1A, 1B, 2, 3A and 4, althoughsamples of genotype 2 and 4 were rarer in our patient collective

All genotypes pooled* 3.568 3700

* No significance between the genotypes (P > 0.05), pooled slopes and intercepts.** Four patients of genotype 3A were excluded from the calculation.

mmunoblots. Various combination of results were found: Anti-CV immunoblots confirmed the presence of anti-HCV antibodies

n 41 cases, implying a resolved hepatitis C infection, or in 6 sam-les carriers of low HCV RNA between 23 and 2620 IU/mL, which iselow the detection limit of the HCV-Ag, respectively. HCV infec-ions were excluded due to unspecific reactivity in 38 (37%) casesy anti-HCV immunoblot, and indeterminate results were found

n 19 samples. On these findings an algorithm is proposed for theonfirmation of first time diagnosed “reactive” anti-HCV (Fig. 4b).

Please cite this article in press as: Ottiger C, et al. Detection limit of architrenewed confirmation algorithm for reactive anti-HCV samples. J Clin Viro

. Discussion

HCV-Ag tests were developed in 1999 [17], but are not yetidely used, although it was shown that active hepatitis C

−2.556

infections were by 24–34 days earlier detected with HCV-Ag thanwith anti-HCV alone [11,14,18]. Automated platforms are ableto perform rapidly anti-HCV and HCV-Ag as single tests in lessthan 15 min, such as Abbott Architect®. However, to analyse twoparameters instead of a combined test adds massively to the costs.The question remains, for which samples both tests should beapplicable: in clinically suspected early hepatitis, for confirmationof reactive anti-HCV antibodies and/or for the follow up of achronic hepatitis C under antiviral treatment?

Our aim was to study the detection limit of the HCV-Ag incomparison with HCV RNA and note eventual differences amongthe genotypes. In addition, a renewed confirmation algorithm wasestablished in cases of first time diagnosed “reactive” anti-HCV.

Our results show a correlation of R2 0.89 between HCV-Ag and

ect hepatitis C core antigen assay in correlation with HCV RNA, andl (2013), http://dx.doi.org/10.1016/j.jcv.2013.08.028

Fig. 2. Dilution experiment of 5 samples, each with a different genotype, for theanalysis of the detection limit of HCV-Ag in comparison with HCV RNA. Legend seeFig. 1.

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Fig. 3. Samples were measured in triplicates over the whole analytical range forHw

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(anti-HCV screening)

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412

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117 (53%)

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but PCR +

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104

221

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b

test anti-HCV

test HCV-Ag

perform anti-HCV

immunoblot

anti-HCV

reactive ?

no HCV-Ag

positive ?

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no hepatitis C(except early � retest in 2 -3 month)

yes

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Fig. 4. Consecutive samples were tested for anti-HCV screening (AHCV scr.) on®

CV-Ag by the Abbott Architect i2000SR® . Coefficient of variation (CV%) of <10%as obtained with HCV-Ag values above the cut-off of 0.477 log10 fmol/L.

Tables 1a and b and Fig. 1). Similar data were found by other [15],ut only samples of genotype 1B were tested; but other studieshowed similar results [6,19,20]. Based on these one might be ableo predict HCV viral load, independent on the genotype. From HCV-g one could extrapolate to HCV RNA levels; this also in few of lowV% of HCV-Ag of <10%, identical with others [15]. Empirically, ouramples were extrapolated by Eq. (1), and calculated RNA were ofn acceptable difference of 0.4 ± 0.37 Log10 IU/mL from the actualiral load. Future multi-centre studies should proof this concept.

At the end of this study Roche introduced a new version 2.0 ofCV RNA on Cobas AmpliPrep/TaqMan®. It had been recognised,

hat version 1.0 underestimated the viral loads of genotype 4 [16],nd much improvements were obtained with version 2.0. There-ore, we retested the samples of genotype 4 with version 2.0, andndeed noted some non systematic differences with version 1.0data not shown). Calculations were thus obtained from version.0.

The detection limit of the HCV-Ag in comparison with HCV RNAas studied, as this is crucial for using this parameter for confir-ation of an active hepatitis C infection, for the follow-up under

herapy as proposed [20], or for the detection of an early hepatitis, where anti-HCV could still be negative [11].

In our collective, it was found that HCV RNA levels were stilletween 2.4 and 4.5 Log10 IU/mL when HCV-Ag became negativeTable 2a), with no significant differences among the genotypesP > 0.5). The dilution experiment of 5 samples, each with a different

Please cite this article in press as: Ottiger C, et al. Detection limit of architrenewed confirmation algorithm for reactive anti-HCV samples. J Clin Viro

enotype, demonstrated similar results with values between 3.34nd 3.74 Log10 IU/mL. Other studies have been conducted wherehe detection limit was in the same range [6,19,20] (Table 3).

able 3CV RNA of 5 EDTA-samples in a diluting experiment at the cut-off value of HCV-Agf 0.477 Log10 fmol/L, i.e. 3 fmol/L.

HCV RNA

Log10 (IU/mL) IU/mL

All genotypes 3.54 3467Genotype 1A 3.571 − 3.729 3724 − 5357Genotype 1B 3.554 − 3.645 3581 − 4416Genotype 2 (A/C) 3.339 − 3.394 2183 − 2477Genotype 3A 3.649 − 3.741 4457 − 5508Genotype 4 (A/C/D) 3.456 − 3.520 2858 − 3311

Abbott Architect i2000SR . In cases of first time diagnosed “reactive” anti-HCV, theywere additionally analysed for HCV-Ag and/or HCV RNA and anti-HCV immunoblot(AHCV blot). (a) Distribution of AHCV scr. and combined results with HCV-Ag, HCVRNA and AHCV-blot are shown (+/− means indeterminate results, and underlined

numbers are from a subgroup). (b) Proposed algorithm for screening and confirma-tion of hepatitis C.

However, HCV-Ag is still a useful parameter for rapid confirma-tion of a “reactive” anti-HCV result, as both are analysed on thesame platform. Positive HCV-Ag confirms thus an active hepatitisC infection, which is the case in about 50% (Fig. 4a). The viral loadcan thus be estimated by linear regression (Eq. (1)), as there is nosignificant difference in titres between the genotypes.

ect hepatitis C core antigen assay in correlation with HCV RNA, andl (2013), http://dx.doi.org/10.1016/j.jcv.2013.08.028

In the case of “reactive” anti-HCV and negative HCV-Ag furthertesting are needed to confirm a resolved hepatitis C infection,or to discover unspecific reactivity of the anti-HCV test. An

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ARTICLECV-2842; No. of Pages 6

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lgorithm is proposed (Fig. 4b). Although a confirmation by anti-CV immunoblot of a “reactive” anti-HCV with negative HCV RNA

s considered obsolete [3], some reflection should be allowed: Asas been shown by our data, one third of “reactive” anti-HCV andegative HCV-Ag were confirmed being false positive (Fig. 4a). This

s indeed “only” 0.25% of all performed tests, but still a considerablemount of all “reactive” samples. False positive serological resultsre often seen in pregnant women, patients with autoimmuneiseases and patients who undergo dialysis. The question woulde posed from which source the hepatitis C infection was acquired,s infections in our area are mainly due to intravenous drug abuse,ransfusion of blood products before 1989, dialysis instruments ormmigrants from an endemic region. The public health authorityf some countries demands notification of all first time confirmedositive anti-HCV, HCV-Ag or HCV RNA [21]. To avoid patients withalse diagnoses of hepatitis C infections, we propose the algorithmFig. 4b) to be followed, similar to the recently published guidance22].

In conclusion HCV-Ag may represent an alternative in lieu ordjunct to HCV RNA determination both for identifying an activeCV infection, and for monitoring antiviral treatement, as long asCV-Ag is still detectable. This is particularly interesting for low

esource countries, because HCV RNA measurements need wellquipped and specialised laboratories, and are quite expensive. Theeasurements of HCV RNA below the cut-off of HCV-Ag is still

eeded to confirm the complete elimination of viruses. Confirma-ory anti-HCV immunoblots are still useful to detect false reactiventi-HCV results by screening tests.

onflict of Interest

None.

unding

None.

ompeting interests

None.

thical approval

Not required.

cknowledgement

We express our thanks to the Department of Laboratoryedicine of the Cantonal Hospital of Lucerne for the quantification

Please cite this article in press as: Ottiger C, et al. Detection limit of architrenewed confirmation algorithm for reactive anti-HCV samples. J Clin Viro

f HCV RNA by Abbott m2000rt® and for the HCV genotyping by theERSANT® method, and to the Laboratory of Allergy and Immunol-gy of the University Hospital of Zurich for anti-HCV recomLinemmunoblots.

[

[

PRESSVirology xxx (2013) xxx– xxx

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