Early Detection of Hepatitis C Virus Infection by Use of a NewCombined Antigen-Antibody Detection Assay: PotentialUse for High-Risk Individuals

A. Schnuriger,1 S. Dominguez,2 M.-A. Valantin,2 R. Tubiana,2 C. Duvivier,2 J. Ghosn,2

A. Simon,3 C. Katlama,2 and V. Thibault1*

Virology,1 Infectious Diseases,2 and Internal Medicine3 Departments, Pitie´-Salpeˆtrie`re Hospital, Paris, France

Received 8 September 2005/Returned for modification 29 October 2005/Accepted 26 January 2006

The performance of a new combined antigen-antibody assay (Monolisa HCV Ag-Ab Ultra; Bio-Rad Laboratories)was evaluated in the context of acute hepatitis C in human immunodeficiency virus-infected patients.The combined assay became positive as early as the first PCR and earlier than a third-generation enzymelinkedimmunosorbent assay in 65% of the cases. Reduction of the long period of HCV seronegativity shouldimprove the diagnosis of hepatitis C infection, especially in high-risk populations.

The early diagnosis of hepatitis C virus (HCV) infection iscrucial to prevent further transmission in high-risk groups andto allow clinicians to make a rapid decision about treatment,which has been proven to have a high degree of efficacy foracute hepatitis C (5). Due to the long period of seronegativity,serological assays (18) remain poorly efficient for diagnosis inthe early stages of HCV infection. Strategies that can be usedto improve the ability to diagnose the infection before seroconversionrequire additional direct tests, such as detection ofviral RNA by PCR or detection of HCV core antigen (Ag).However, genomic tests, which are limited by the requirementfor specific equipment, a long delay in the time to a result, the

risk of contamination, and high costs, cannot be used as routinescreening tools. Moreover, the assay based on detection ofthe HCV core protein (Trak-C; Ortho Clinical Diagnostics)(12) and proposed as an alternative to PCR (3, 19) is no longeravailable in several countries and suffered from a lack of sensitivityand relatively long hands-on time. The simultaneousdetection of both antigen and antibody (Ab) is now commonlyused for the diagnosis of human immunodeficiency virus(HIV) infection, with the result being a dramatic shortening ofthe so-called seronegative window and, subsequently, the earlierdiagnosis of recent infection. A similar approach based onthe HCV core protein and specific anti-HCV antibody detection

(Monolisa HCV Ag-Ab Ultra; Bio-Rad Laboratories,Marnes-la-Coquette, France) has recently been developed forthe diagnosis of hepatitis C. The overall performance of thiscombined assay has previously been evaluated with seroconversionpanels and HCV RNA-positive HCV Ab-negative

samples (2, 10, 11).

In the context of HIV infection, the HCV seroconversionelay is often prolonged (16), and the failure of reactivity ofcurrent HCV Ab detection tests (9, 15) often requires PCR forHCV RNA detection. The aim of this study was to completeprevious evaluations of the Monolisa HCV Ag-Ab Ultra assay(2, 10, 11) by assessing its potential clinical benefit in thesetting of acute HCV infection in HIV-infected patients.

   Twenty HIV-infected patients were recently identified tohave acute hepatitis C. These patients were among the patientsin our local cohort of HIV-infected homosexual males withhigh-risk sexual behaviors (6). The patients had been HIVinfected for 5 to 19 years, all were receiving highly activeantiretroviral therapy, and for most of them, the HIV infectionwas well controlled. Stored plasma samples were availablefrom quarterly HIVviral load monitoring. Hepatitis C was firstsuspected by an increase in the alanine transaminase (ALT)level and/or the personal history of the patient and was furtherconfirmed by two consecutive positive HCV RNA detections.HCV infection was retrospectively proven by the use of storedfrozen serum and plasma samples: all cases were previouslyHCV seronegative and HCV RNA negative by PCR (COBASAMPLICOR HCV Monitor test, version 2.0; Roche Diagnostics,Meylan, France) and later showed a positive result forHCV RNA, followed by seroconversion (detected by theMonolisa anti-HCV Plus V2 assay; Bio-Rad) for all but one ofthe patients. HCV genotypes were determined by sequencingof the NS5B region (17). The level of total HCV core antigenwas measured by using the Trak-C immunoassay (Ortho ClinicalDiagnostics). Samples were analyzed by the MonolisaHCV Ag-Ab Ultra assay (Bio-Rad), according to the manufacturer’srecommendations. The results are expressed as theratio of the absorbance to the cutoff; a ratio above 1 is considereda positive result. As suggested previously, we also considered

a sensitized threshold (gray zone) at a ratio of 0.5 (2,11).

   We first confirmed with a cohort of 160 HIV-infected patients(80 were HCV seronegative and 80 were chronically coinfectedwith HCV and HIV) the overall excellent specificity and sensitivityof the Monolisa HCV Ag-Ab Ultra assay (data not shown). Inthis cohort, the proposed sensitized threshold ratio of 0.5 wasabove the mean value (0.011) for the seronegative samples plus 10standard deviations.Ninety-four samples from our panel of samples from 20HIV-seropositive patients with acute HCV infections weretested by the combined Monolisa HCV Ag-Ab assay in parallel

TABLE 1. HIV and HCV infection characteristics and performances of the Ag-Ab assay expressed as delays of HCV infectiondetection compared to those by reference methods图表见附件:HCVAg表格

with HCV RNA detection and anti-HCV Ab tests (Table 1).Detection by the Monolisa HCV Ag-Ab assay occurred earlierthan by the Ab assay for 13 patients (65%). The mean delay ofHCV infection detection by the new test was 27 days after thefirst positive result for HCV RNA detection (and 9 days whenthe optimized threshold of 0.5 was used), whereas it was 77days by Ab detection alone. In our specific population, the Abdetection assay would then become positive on average 50 daysafter the Ag-Ab assay (and 68 days by use of the optimizedthreshold of 0.5). In 65% of the cases, the combined assaybecame positive with the same blood sample used for HCVRNA detection. With the optimized threshold of 0.5, reactivityat the time of HCV RNA detection was detected in 85% of thecases. Interestingly, for most patients (9 of 12) for whom follow-up ALT levels were available, the first detection of HCVinfection by the combined assay occurred before the peak ALTlevel was detected (data not shown).

For patient 11, who had a prolonged seronegative period over18 months, a lack of reactivity by the Ag-Ab assay was observed,despite high HCV viral loads (over 1 million IU/ml) in severalconsecutive samples. It is noteworthy that values between 0.5 and1 were obtained for most samples. The core coding gene sequencewas determined in two samples from this patient by directsequencing: the core protein was 98% identical to theGI:532369 GenBank sequence, and no major change in thesequence from those of other genotype 4 core proteins wasnoticed (4). The core protein level in several samples wasmeasured by the Ortho Trak-C assay and ranged from 39 to406 pg/ml, which are values in accordance with the viral loadlevels (3). The production of antibody against each HCV proteinwas analyzed by an immunoblot assay, which eventuallybecame positive.

Diagnosis of acute hepatitis C largely relies on classicalserological methods (1). However, the value of such Ab detectionassays is limited during the early stages of HCV infection,first, because specific antibodies develop slowly and, second,because immunocompromised patients may fail to develop astrong and rapid specific immune response against HCV. Thedevelopment of new assays that combine both Ag and Abdetection, similar to what has been done in the field of HIVdetection (13), may prove useful in reducing the long windowof HCV seronegativity or compensating for the absence of aspecific antibody response. This is particularly true in the caseof HIV coinfection, where long periods of seroconversion havebeen described (14, 16). Those combined tests present a potentialadvantage and may improve commonly used serologicalassays without the need to resort to an additional direct detectionprocedure, such as PCR or Ag measurement.

In this evaluation of the Monolisa HCV Ag-Ab Ultra assaywith samples from 20 HIV-infected patients who developed acuteHCV infection, the new combined test allowed, in most cases, thedetection of hepatitis C infection earlier than that by conventionalserological assays. Obviously, calculation of delays between thereactivity of the serological assay and that of the combined assaywould be biased in this study due to heterogeneous intervalsbetween the times of testing of two consecutive samples from apatient and the long HCV-seronegative period usually observedinHIV-infected patients. Nevertheless, these long intervals arethose observed in the “real” clinical setting and in the managementof HIV-infected patients. Of note, our detection delays areconsistent with those previously calculated with either commercial(30 days) or so-called natural (28 days) seroconversion panels

(2, 10). Importantly, for a majority of patients, the combined assaybecame positive as early as HCV RNA detection. In our population,the combined assay never failed at identifying a seroconversionthat would have been detected by the Monolisa anti-HCVPlus assay.

Unexpectedly, for one patient who showed persistent HCVreplication but an absence of seroconversion for more than 1year, the combined assay led to only a weak response. Sequencingof the core encoding gene and quantification of the coreprotein by the Trak-C assay did not support any peculiarity ofthe HCV core protein, and reactivity by immunoblot testingwas evidenced for this patient. Thus, the weak reactivity wouldreflect in this case a lack of sensitivity of the Monolisa HCVAg-Ab Ultra assay for the detection of the core protein andanti-HCV Ab. Other hypotheses should be investigated, however,like presence of immune complexes or the interaction ofthe virus with lipid components. The low reactivity observed

for this patient also reinforces the usefulness of establishing agray zone ratio of 0.5 to 1 that may permit the better identificationof samples in the seroconversion window, as previouslysuggested in two studies (2, 11). The sensitivity of Ag detectionby the combined test might be less satisfying compared to thatby HCV core Ag detection assays. However, the purpose ofthis new combined test is mainly to improve and eventuallyreplace the serological assays used as first-line screening tools,whereas core Ag detection assays would rather be used as areplacement for nucleic acid testing (7, 8).

   In conclusion, although the influence of the HIV infectionstatus on HCV detection by the Monolisa HCV Ag-Ab Ultraassay needs to be confirmed with larger panels of samples,including samples from very immunocompromised patients,the results of this first study with samples from HIV-infected

patients are promising. Similar to what has been demonstratedfor the Ag-Ab combined assays used for the diagnosis of HIVinfection, the use of such a combined assay for HCV detectionbrings a real clinical benefit compared to conventional serologicalassays by shortening the seronegative window period.Such an improvement may be particularly useful for the diagnosisof HCV infection in high-risk groups, where early infectiondetection may help reduce secondary transmissions andrapid referral for treatment. Because of its relatively low cost,its ease of performance, and the significant decreased windowperiod, we believe that this new assay will improve theoverall quality of diagnosis of HCV infection for a moderateoverall cost compared to those of serological assays andshould therefore be applied as a screening assay in thegeneral population.

     We must emphasize that this assay is not intended to replaceHCV RNA detection, and in the case of acute clinical hepatitis,the use of a sensitive HCV RNA detection assay is highlyrecommended. Because the sensitivity of the HCV core detectionassay is significantly lower than that of any PCR-basedmethod, it should not be recommended for use for this particularpurpose.

A.S. is supported by a grant from Sidaction. B. Slizewicz from Bio-Rad Laboratories kindly provided Monolisa HCV Ag-Ab Ultra reagents.We are grateful to S. Laperche for her scientific expertise and HCVcore Ag measurement.

REFERENCES

1. Alter, M. J., W. L. Kuhnert, and L. Finelli. 2003. Guidelines for laboratorytesting and result reporting of antibody to hepatitis C virus. Morb. Mortal.Wkly. Rep. Recomm. Rep. 52:1–13, 15.

2. Ansaldi, F., B. Bruzzone, G. Testino, M. Bassetti, R. Gasparini, P. Crovari, andG. Icardi. 2006. Combination hepatitis C virus antigen and antibody immunoassayas a new tool for early diagnosis of infection. J. Viral Hepat. 13:5–10.

3. Bouvier-Alias, M., K. Patel, H. Dahari, S. Beaucourt, P. Larderie, L. Blatt, CHezode, G. Picchio, D. Dhumeaux, A. U. Neumann, J. G. McHutchison, andJ. M. Pawlotsky. 2002. Clinical utility of total HCV core antigen quantification:a new indirect marker of HCV replication. Hepatology 36:211–218.

4. Bukh, J., R. H. Purcell, and R. H. Miller. 1994. Sequence analysis of the coregene of 14 hepatitis C virus genotypes. Proc. Natl. Acad. Sci. USA 91:8239–8243.

5. Gerlach, J. T., H. M. Diepolder, R. Zachoval, N. H. Gruener, M. C. Jung, A.Ulsenheimer, W. W. Schraut, C. A. Schirren, M. Waechtler, M. Backmund,and G. R. Pape. 2003. Acute hepatitis C: high rate of both spontaneous andtreatment-induced viral clearance. Gastroenterology 125:80–88.

6. Ghosn, J., S. Pierre-Francois, V. Thibault, C. Duvivier, R. Tubiana, A. Simon,M. A. Valantin, S. Dominguez, E. Caumes, and C. Katlama. 2004. Acute hepatitisC in HIV-infected men who have sex with men. HIV Med. 5:303–306.

7. Icardi, G., F. Ansaldi, B. M. Bruzzone, P. Durando, S. Lee, C. de Luigi, andP. Crovari. 2001. Novel approach to reduce the hepatitis C virus (HCV)window period: clinical evaluation of a new enzyme-linked immunosorbentassay for HCV core antigen. J. Clin. Microbiol. 39:3110–3114.

8. Krajden, M., R. Shivji, K. Gunadasa, A. Mak, G. McNabb, M. Friesenhahn,D. Hendricks, and L. Comanor. 2004. Evaluation of the core antigen assay asa second-line supplemental test for diagnosis of active hepatitis C virusinfection. J. Clin. Microbiol. 42:4054–4059.

9. Lamoril, J., F. Lunel, P. Laurent-Puig, C. Defer, P. Loiseau, J. J. Lefrere,J. M. Pawlotsky, P. Marcellin, F. Bouchardeau, M. Bogard, et al. 1994.Indeterminate third-generation recombinant immunoblot assay in hepatitisC virus infection. J. Hepatol. 21:133–134.

10. Laperche, S., M.-H. Elghouzzi, P. Morel, M. Asso-Bonnet, N. Le Marrec, A.Girault, A. Servant-Delmas, F. Bouchardeau, M. Deschaseaux, and Y. Piquet.2005. Is an assay for simultaneous detection of hepatitis C virus core antigenand antibody a valuable alternative to nucleic acid testing? Transfusion45:1965–1972.

11. Laperche, S., N. Le Marrec, A. Girault, F. Bouchardeau, A. Servant-Delmas,M. Maniez-Montreuil, P. Gallian, T. Levayer, P. Morel, and N. Simon. 2005.Simultaneous detection of hepatitis C virus (HCV) core antigen and anti-HCV antibodies improves the early detection of HCV infection. J. Clin.Microbiol. 43:3877–3883.

12. Laperche, S., N. Le Marrec, N. Simon, F. Bouchardeau, C. Defer, M.Maniez-Montreuil, T. Levayer, J. P. Zappitelli, and J. J. Lefrere. 2003. Anew HCV core antigen assay based on disassociation of immune complexes:an alternative to molecular biology in the diagnosis of early HCV infection.Transfusion 43:958–962.

13. Ly, T. D., S. Laperche, C. Brennan, A. Vallari, A. Ebel, J. Hunt, L. Martin,D. Daghfal, G. Schochetman, and S. Devare. 2004. Evaluation of the sensitivityand specificity of six HIV combined p24 antigen and antibody assays.J. Virol. Methods 122:185–194.

14. Morand, P., N. Dutertre, H. Minazzi, J. Burnichon, M. Pernollet, M. Baud,J. P. Zarski, and J. M. Seigneurin. 2001. Lack of seroconversion in a healthcare worker after polymerase chain reaction-documented acute hepatitis Cresulting from a needlestick injury. Clin. Infect. Dis. 33:727–729.

15. Pawlotsky, J. M., A. Bastie, C. Pellet, J. Remire, F. Darthuy, L. Wolfe, C.Sayada, J. Duval, and D. Dhumeaux. 1996. Significance of indeterminatethird-generation hepatitis C virus recombinant immunoblot assay. J. Clin.Microbiol. 34:80–83.

16. Ridzon, R., K. Gallagher, C. Ciesielski, M. B. Ginsberg, B. J. Robertson,C. C. Luo, and A. DeMaria, Jr. 1997. Simultaneous transmission of humanimmunodeficiency virus and hepatitis C virus from a needle-stick injury.N. Engl. J. Med. 336:919–922.

17. Sandres-Saune, K., P. Deny, C. Pasquier, V. Thibaut, G. Duverlie, and J.Izopet. 2003. Determining hepatitis C genotype by analyzing the sequence of

the NS5b region. J. Virol. Methods 109:187–193.

18. Tobler, L. H., S. L. Stramer, S. R. Lee, B. L. Masecar, J. E. Peterson, E. A.Davis, W. E. Andrews, J. P. Brodsky, S. H. Kleinman, B. H. Phelps, andM. P. Busch. 2003. Impact of HCV 3.0 EIA relative to HCV 2.0 EIA onblood-donor screening. Transfusion 43:1452–1459.

19. Veillon, P., C. Payan, G. Picchio, M. Maniez-Montreuil, P. Guntz, and F.Lunel. 2003. Comparative evaluation of the total hepatitis C virus coreantigen, branched-DNA, and AMPLICOR MONITOR assays in determiningviremia for patients with chronic hepatitis C during interferon plusribavirin combination therapy. J. Clin. Microbiol. 41:3212–3220.