Comparison of Serum Hepatitis C Virus (HCV) RNA an
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微生物临床杂志 2006年第2期
ABSTRACT
Trak-C (Ortho-Clinical Diagnostics) is an enzyme-linked immunosorbent assay-based method capable of quantifying hepatitis C virus (HCV) core antigen (CA) in serum and could be an alternative to molecular detection and quantification of HCV RNA. We have evaluated the Trak-C assay in comparison with an HCV RNA quantitative assay (Versant HCV v3.0; Bayer Diagnostics) in the follow-up of 348 treated, human immunodeficiency virus (HIV)/HCV-coinfected patients included in the ANRS HC02 RIBAVIC trial. ANRS HC02 RIBAVIC is a therapeutic, multicenter, randomized protocol comparing the efficacy of alpha interferon 2b (IFN-2b) (3 million units three times a week)-ribavirin (800 mg/day) to that of pegylated IFN-2b (1.5 μg/kg of body weight/week)-ribavirin (800 mg/day) during 48 weeks of treatment of HIV/HCV-coinfected patients na?ve to HCV treatment. Patients were assessed for virological analysis at day 0 and weeks 4, 12, 24, 48, and 72. Correlation of HCV RNA and HCV CA at the initiation of treatment was excellent (r = 0.92). HCV RNA and CA kinetics were similar during follow-up of HCV treatment from day 0 to week 72 whatever the group of response and genotype. The positive and negative predictive values of response to the treatment at week 4 were 59 and 94%, respectively, for HCV RNA load reduction of >2 log and 54 and 94%, respectively, for HCV CA below the threshold value (4.18 log10 pg/ml · 104). Trak-C, a new assay able to quantify CA in HIV/HCV-coinfected patients, correlates well with quantitative HCV RNA assays and is cheaper and easier to perform than molecular technology. HCV CA could be a valuable alternative test for therapeutic follow-up of coinfected patients treated with IFN plus ribavirin in developing countries.
INTRODUCTION
Today, RNA detection and quantification are the only systems for pretherapeutic and therapeutic follow-up of hepatitis C virus (HCV)-infected persons undergoing treatment (4, 6, 8, 11, 17, 23). Measures of HCV RNA before treatment and at 12 weeks of treatment are used to determine early decrease of viral load during treatment. A 2-log HCV RNA variation is considered a reference for management of treatment for HCV-infected persons (1, 14). Since RNA detection is labor-intensive and very expensive, many laboratories have sought to replace HCV RNA detection or quantification by other markers. Moreover, HCV core antigen (CA) is more stable than HCV RNA and needs no particular precautions for preparation and sample storage (21). Several kits for detection of HCV CA have recently been developed and commercialized (19). Methods for detecting HCV CA using monoclonal antibody to HCV CA were also developed, such as the Trak-C assay, developed by Ortho-Clinical Diagnostics and commercialized in 2003 (21). However, the low sensitivity of this assay needs to be evaluated in pretherapeutic and therapeutic follow-up of HCV-infected patients. This enzyme-linked immunosorbent assay (ELISA)-based method has already been demonstrated with HCV-infected patients as a new diagnosis marker for HCV infection, with an excellent correlation observed for all studies (9, 12, 21, 22, 24, 25).
The framework of the ANRS HC02 RIBAVIC protocol included 412 human immunodeficiency virus (HIV)/HCV-coinfected patients and demonstrated the efficacy and safety of the anti-HCV combined interferon (IFN)-ribavirin therapy (3). In this study, viral factors of treatment response prediction were identified: a baseline viremia below 5.7 log10 IU/ml and genotypes 2, 3, and 5 (as reported by others [2, 5]).
Therefore, we have evaluated the usefulness of the new HCV CA assay (Trak-C assay; Ortho-Clinical Diagnostics) in comparison with the HCV RNA load for therapeutic follow-up of HIV/HCV-coinfected patients included in the ANRS HC02 RIBAVIC study. We have shown that the HCV CA assay assessed virological response similarly to the HCV RNA assay and had a good predictive value of nonresponse to combination therapy.
MATERIALS AND METHODS
Patient study: the ANRS HC02 RIBAVIC protocol. For the comparison of HCV RNA and CA evolution, we tested approximately 1,500 samples from the follow-up of the first 348 HIV/HCV-coinfected patients included in the ANRS HC02 RIBAVIC protocol from February 2000 to February 2002 (n = 412), performed in 71 French centers. This protocol is a multicenter, randomized, parallel-group, open-label trial comparing two arms: alpha interferon 2b (IFN-2b) at 3 million units three times a week (n = 207) or pegylated IFN-2b at 1.5 μg/kg of body weight/week (n = 205) with (in each arm) ribavirin 800 mg/day over 48 weeks (3). Samples for viral follow-up were collected at day (D) 0 and weeks (W) 4, 12, 24, 36, and 48, centralized, and kept at –80°C in two laboratories (CHU Angers and CHU Grenoble, France). Follow-up evaluations were finished 24 (W72) weeks after the end of treatment. This was the main endpoint to assess sustained virological response (undetectable HCV RNA at W72). Four groups of patients were considered according to their response to treatment: group 1, nonresponders (NR), with persistent HCV RNA under treatment (n = 198, 56.9%); group 2, sustained virological responders (SVR), with undetectable HCV RNA under treatment and 6 months after stopping treatment (n = 93, 26.7%); group 3, relapsers (RR), with undetectable HCV RNA under treatment and detectable HCV RNA at W72 (n = 25, 7.2%); and group 4, responder patients with a "breakthrough" (RB), defined as undetectable HCV RNA under treatment and reappearance of RNA before the end of treatment despite continuation of therapy (n = 32, 9.2%). In fact, we cannot certify perfect observance for all of these breakthrough patients. Patients from groups 1, 3, and 4 were considered non-SVR for comparison with group 2 in this study.
HCV RNA quantification. Quantification of HCV RNA (50 μl) was done with HCV RNA positive samples, determined by Cobas Amplicor HCV v2.0 (Roche Diagnostics, Meylan, France). Serum samples were tested at D0 and W4, 12, 24, 36, 48, and 72 by branched-DNA technology, using Versant HCV RNA v3.0 according to the manufacturer's instructions (Bayer Diagnostics, Eragny, France). Results were expressed in log10 IU/ml. The range of HCV RNA quantification is from 2.79 log10 IU/ml to 6.89 log10 IU/ml.
Quantitative detection of total HCV core antigen. Samples tested for quantification of HCV RNA were also assessed for total HCV CA by the Trak-C assay (Ortho-Clinical Diagnostics, Les Ulis, France). This novel assay is an ELISA-based method for microplate using 100 μl of serum performed according to the manufacturer's instructions. The range of detection is approximately 1.5 pg/ml to 300 pg/ml (about 4 to 6.4 log10 IU/ml).
HCV genotype determination. Patient samples were genotyped at D0 for all patients by using the Versant HCV genotype assay (Bayer Diagnostics) according to the manufacturer's instructions. This assay was done with 20 μl of amplicons from Cobas Amplicor technology.
Statistical analysis. The statistical analysis was performed with SPSS software version 10.1 for Windows (Statistical Package of Service Solutions; SPSS Inc., Chicago, Ill.). Results from CA quantification were corrected with a 104 factor and expressed in log10 (pg/ml · 104) for the correlation study with RNA quantification (log10 IU/ml). When quantification results (HCV RNA and CA) were under the cutoff value, we used the logarithm of half the cutoff value [2.49 log10 IU/ml for RNA and 3.88 log10 (pg/ml · 104) for CA]. The correlation study was done by Pearson's test. All statistical significance was assessed at the level where P was 0.05.
RESULTS
Study of the two markers at initiation of treatment. A very good correlation between HCV RNA and CA levels of the 348 patients at initiation of treatment (D0) was found (0.92, P < 0.01) (Fig. 1). This correlation at initiation of treatment was observed independently of type of response and HCV genotype (Table 1). Intra- and intergenotype sensitivity studies indicated no significant difference between HCV RNA and HCV CA for the four most frequent HCV genotypes (1, 2, 3, and 4) (Table 1 and Fig. 2).
Viral kinetics sorted by response to treatment. We obtained similar kinetics for the two viral markers, HCV RNA and HCV CA, for each type of per-protocol patient response when taking into account the different cutoff values of these assays (2.79 log10 RNA and 4.18 log10 CA) (Fig. 3). So, when the optical density is under the cutoff value, curves are not similar. Viral kinetics in nonresponders showed a mean transient decrease of about 1 log10 between D0 and W12 for both markers (1.07 ± 1.13 log10 RNA and 0.96 ± 0.86 log10 CA). In sustained responders, the early decrease (W4) of RNA and CA (3.12 ± 1.18 and 1.75 ± 1.02, respectively) was significantly different than that of relapsers (2.03 ± 1.22 and 1.68 ± 1.06, respectively; P < 0.001); the latter was also significantly different than that of nonresponders (0.83 ± 0.89 and 0.81 ± 0.74, respectively; P < 0.001). For patients presenting a breakthrough during treatment, decrease at W4 (2.49 ± 1.14 log10 RNA and 1.73 ± 0.86 log10 CA) was significantly different from that for RR and SVR (P < 0.01) (Table 2).
Predictive values of response and nonresponse. The negative predictive value (NPV) and the positive predictive value (PPV) of response to treatment were calculated according to HCV RNA or HCV CA below the cutoff value or to a decrease of HCV RNA or HCV CA superior than 2 log10 under treatment. NPV and PPV were studied at W4 and W12 (Table 3). The best PPV was observed using undetectable HCV RNA at W4 (72%). Finally, 71% of SVR had undetectable HCV RNA at W4. The best NPV was obtained at W12 using undetectable HCV CA alone as well as undetectable HCV RNA or a 2-log reduction for HCV RNA (99% for both parameters), allowing detection of 65% or 64% of NR, respectively.
DISCUSSION
In this study, we have evaluated for the first time the use of a new HCV marker (HCV CA) assay in comparison with a quantitative HCV RNA assay for baseline viral load and early virological response in HCV/HIV-coinfected patients included in the ANRS HC02 RIBAVIC study. It has been recommended from large pivotal trials (13, 16) to evaluate the early virological response after 12 weeks of treatment, based on a 2-log decline or a negative viral RNA algorithm (1, 14). This approach allows early discontinuation of treatment for patients who will not respond and, thus, could avoid side effects and expense for these patients. General application of this algorithm is dependent on HCV RNA quantitative methods, which are cost-effective and not accessible in every laboratory for patient follow-up.
Many studies have observed a very good correlation (0.72 < r < 0.80) between quantification methods of HCV RNA and HCV CA in HCV-monoinfected patients (7, 12, 20, 22, 24). In HCV/HIV-coinfected patients, we also found an excellent correlation at baseline between these two markers of HCV infection (0.92). In our study, 15 patients (4.4%) had an undetectable HCV CA at baseline, with detectable HCV RNA between 615 and 101,447 IU/ml (mean of 18,417 IU/ml). Soffredini et al. observed that 6% of HCV-monoinfected patients (7/111) had undetectable HCV CA with detectable RNA at initiation of IFN or pegylated IFN plus ribavirin treatment. The threshold of HCV CA quantification was also evaluated at 15,000 IU/ml (20). It is important to underline that only 1 patient among the 15 patients found to be CA negative and RNA positive at baseline was a nonresponder. Sensitivity of the HCV CA quantification may be increased with automated technology, such as using chemiluminescent detection with the Lumipulse HCV Ortho antigen assay (21). The range of HCV CA detection using Lumipulse is 1 to 1,000 pg/ml, whereas the Trak-C range is 1.5 to 100 pg/ml, and correlation with HCV RNA for Lumipulse is 0.87 (21). In our previous study of HCV RNA and CA correlation in HCV-monoinfected patients, we showed that 1 pg/ml of HCV CA was approximately equivalent to 10,000 IU/ml, as confirmed by Massaguer et al. (12). In HIV/HCV-coinfected patients, we observed a correspondence of 8,400 IU/pg at D0.
During IFN-ribavirin treatment, kinetics of the two HCV markers were similar (when optical density was above the cutoff value for both assays), whatever the arm of treatment, genotype, or HIV infection characteristics, as we observed with HCV-infected patients (24). Two early phases of viral elimination were observed with SVR, as previously described for HCV-infected, treated patients: a very early phase with a rapid viral decline, before W4, and a second phase with a slower decline, between W4 and W12 (15, 26). In RR, the first phase of decrease was slower than that of SVR and was prolonged up to W12. Indeed, it was shown that the rate of the phase 2 decay correlated closely with that of SVR to IFN-based treatment regimens (26). Especially for NR, a very weak decline was observed before W12. A viral rebound was always observed after W12, with return to baseline value. Unlike the results reported by Schüttler et al., our studies did not demonstrate any variations of HCV RNA and HCV CA ratio whatever the status of treatment (before or during treatment) (18). It was hypothesized that HCV RNA-free particles containing HCV CA were produced during treatment and secreted by infected cell or generated by in vivo degradation of HCV (18).
In the context of HIV/HCV-coinfected patients, early virological response is generally defined as an undetectable HCV RNA at W12 of treatment or as a 2 log10 decrease from baseline of HCV RNA level, as is defined for HCV-monoinfected patients (1, 5, 14). We have studied the PPV and NPV for several parameters, including undetectable HCV CA. We observed that the best NPV (99%) was obtained at W12 using three parameters individually, as described for HCV-monoinfected patients: undetectable HCV CA, significant decrease from D0 to W12 of HCV RNA, and significant decrease from D0 to W12 or undetectable HCV CA (10) (independently of genotype or arm of treatment). By contrast, a decrease of HCV CA of >2 log from D0 to W12 did not predict SVR because of the high threshold of this assay.
We have previously shown that the new HCV CA assay (Trak-C) is a valuable test, well correlated with the HCV RNA assay in the follow-up of patients with chronic hepatitis C treated with IFN plus ribavirin. This ELISA-based method is also cheaper (cost is 73% lower than that of quantitative PCR, as reported for Lumipulse) and easier to perform than molecular tests. However, its sensitivity needs to be improved, particularly in the assessment of the final response, as it is not sensitive enough to measure the sustained virological response (21). Chemiluminescence detection, as recently demonstrated, may increase the sensitivity of the assay as much as twofold (21). Moreover, the stability of HCV core antigen in clinical samples is higher than that of RNA (21).
In conclusion, we showed in this study that, for HCV/HIV coinfected patients, (i) Trak-C is well correlated with the HCV RNA assay whatever the HCV genotype and (ii) HCV CA could be an alternative to HCV RNA for therapeutic follow-up. Thus, the Trak-C core antigen assay could be useful as a low-priced alternative test to nucleic acid detection, particularly in developing countries.
ACKNOWLEDGMENTS
ANRS (French National Agency for Research on AIDS and Viral Hepatitis) was the sponsor of the ANRS HC02 RIBAVIC study, which was conducted with the support of Schering Plough (C. Lemonnier and A. Rimailho).
We thank all of the investigators and virologists involved in the ANRS HC02 RIBAVIC study in France (F. Bani-Sadr [Groupe Hospitalier Universitaire Est, Université Paris 6, Inserm U444], E. Rosenthal [H?pital de l'Archet, Faculté de médecine, Nice], A. Benzeckri and C. Degott [Groupe Hospitalier Universitaire Nord, Université Paris 7], C. Goujard [Groupe Hospitalier Universitaire Sud, Université Paris 11], G. Pialoux [Groupe Hospitalier Universitaire Est, Université Paris 6], L. Piroth [Centre Hospitalier Universitaire, Dijon], and D. Salmon-Céron [Groupe Hospitalier Universitaire Ouest, Université Paris 5]) and D. Lesteven from Ortho-Clinical Diagnostics, France, for supplying the Trak-C kits. Many thanks to Kevin L. Erwin, qualified biomedical translator, for proofreading the manuscript.
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Laboratoire de Bactériologie-Virologie-Hygiène Hospitalière, CHU Angers, France
1 Laboratoire de Virologie, CHU Grenoble, France
2 Inserm U707, Faculté de Médecine St Antoine, Paris, France
3 Service d’Hépatologie, H?pital Necker-Enfants Malades, Paris, France
4 Service de Médecine Interne, H?pital Pitié-Salpétrière, Paris, France
5 Service des Maladies Infectieuses et Tropicales, Hopital Raymond Poincaré, Paris, France(A. Pivert,C. Payan, P. Mo)
Trak-C (Ortho-Clinical Diagnostics) is an enzyme-linked immunosorbent assay-based method capable of quantifying hepatitis C virus (HCV) core antigen (CA) in serum and could be an alternative to molecular detection and quantification of HCV RNA. We have evaluated the Trak-C assay in comparison with an HCV RNA quantitative assay (Versant HCV v3.0; Bayer Diagnostics) in the follow-up of 348 treated, human immunodeficiency virus (HIV)/HCV-coinfected patients included in the ANRS HC02 RIBAVIC trial. ANRS HC02 RIBAVIC is a therapeutic, multicenter, randomized protocol comparing the efficacy of alpha interferon 2b (IFN-2b) (3 million units three times a week)-ribavirin (800 mg/day) to that of pegylated IFN-2b (1.5 μg/kg of body weight/week)-ribavirin (800 mg/day) during 48 weeks of treatment of HIV/HCV-coinfected patients na?ve to HCV treatment. Patients were assessed for virological analysis at day 0 and weeks 4, 12, 24, 48, and 72. Correlation of HCV RNA and HCV CA at the initiation of treatment was excellent (r = 0.92). HCV RNA and CA kinetics were similar during follow-up of HCV treatment from day 0 to week 72 whatever the group of response and genotype. The positive and negative predictive values of response to the treatment at week 4 were 59 and 94%, respectively, for HCV RNA load reduction of >2 log and 54 and 94%, respectively, for HCV CA below the threshold value (4.18 log10 pg/ml · 104). Trak-C, a new assay able to quantify CA in HIV/HCV-coinfected patients, correlates well with quantitative HCV RNA assays and is cheaper and easier to perform than molecular technology. HCV CA could be a valuable alternative test for therapeutic follow-up of coinfected patients treated with IFN plus ribavirin in developing countries.
INTRODUCTION
Today, RNA detection and quantification are the only systems for pretherapeutic and therapeutic follow-up of hepatitis C virus (HCV)-infected persons undergoing treatment (4, 6, 8, 11, 17, 23). Measures of HCV RNA before treatment and at 12 weeks of treatment are used to determine early decrease of viral load during treatment. A 2-log HCV RNA variation is considered a reference for management of treatment for HCV-infected persons (1, 14). Since RNA detection is labor-intensive and very expensive, many laboratories have sought to replace HCV RNA detection or quantification by other markers. Moreover, HCV core antigen (CA) is more stable than HCV RNA and needs no particular precautions for preparation and sample storage (21). Several kits for detection of HCV CA have recently been developed and commercialized (19). Methods for detecting HCV CA using monoclonal antibody to HCV CA were also developed, such as the Trak-C assay, developed by Ortho-Clinical Diagnostics and commercialized in 2003 (21). However, the low sensitivity of this assay needs to be evaluated in pretherapeutic and therapeutic follow-up of HCV-infected patients. This enzyme-linked immunosorbent assay (ELISA)-based method has already been demonstrated with HCV-infected patients as a new diagnosis marker for HCV infection, with an excellent correlation observed for all studies (9, 12, 21, 22, 24, 25).
The framework of the ANRS HC02 RIBAVIC protocol included 412 human immunodeficiency virus (HIV)/HCV-coinfected patients and demonstrated the efficacy and safety of the anti-HCV combined interferon (IFN)-ribavirin therapy (3). In this study, viral factors of treatment response prediction were identified: a baseline viremia below 5.7 log10 IU/ml and genotypes 2, 3, and 5 (as reported by others [2, 5]).
Therefore, we have evaluated the usefulness of the new HCV CA assay (Trak-C assay; Ortho-Clinical Diagnostics) in comparison with the HCV RNA load for therapeutic follow-up of HIV/HCV-coinfected patients included in the ANRS HC02 RIBAVIC study. We have shown that the HCV CA assay assessed virological response similarly to the HCV RNA assay and had a good predictive value of nonresponse to combination therapy.
MATERIALS AND METHODS
Patient study: the ANRS HC02 RIBAVIC protocol. For the comparison of HCV RNA and CA evolution, we tested approximately 1,500 samples from the follow-up of the first 348 HIV/HCV-coinfected patients included in the ANRS HC02 RIBAVIC protocol from February 2000 to February 2002 (n = 412), performed in 71 French centers. This protocol is a multicenter, randomized, parallel-group, open-label trial comparing two arms: alpha interferon 2b (IFN-2b) at 3 million units three times a week (n = 207) or pegylated IFN-2b at 1.5 μg/kg of body weight/week (n = 205) with (in each arm) ribavirin 800 mg/day over 48 weeks (3). Samples for viral follow-up were collected at day (D) 0 and weeks (W) 4, 12, 24, 36, and 48, centralized, and kept at –80°C in two laboratories (CHU Angers and CHU Grenoble, France). Follow-up evaluations were finished 24 (W72) weeks after the end of treatment. This was the main endpoint to assess sustained virological response (undetectable HCV RNA at W72). Four groups of patients were considered according to their response to treatment: group 1, nonresponders (NR), with persistent HCV RNA under treatment (n = 198, 56.9%); group 2, sustained virological responders (SVR), with undetectable HCV RNA under treatment and 6 months after stopping treatment (n = 93, 26.7%); group 3, relapsers (RR), with undetectable HCV RNA under treatment and detectable HCV RNA at W72 (n = 25, 7.2%); and group 4, responder patients with a "breakthrough" (RB), defined as undetectable HCV RNA under treatment and reappearance of RNA before the end of treatment despite continuation of therapy (n = 32, 9.2%). In fact, we cannot certify perfect observance for all of these breakthrough patients. Patients from groups 1, 3, and 4 were considered non-SVR for comparison with group 2 in this study.
HCV RNA quantification. Quantification of HCV RNA (50 μl) was done with HCV RNA positive samples, determined by Cobas Amplicor HCV v2.0 (Roche Diagnostics, Meylan, France). Serum samples were tested at D0 and W4, 12, 24, 36, 48, and 72 by branched-DNA technology, using Versant HCV RNA v3.0 according to the manufacturer's instructions (Bayer Diagnostics, Eragny, France). Results were expressed in log10 IU/ml. The range of HCV RNA quantification is from 2.79 log10 IU/ml to 6.89 log10 IU/ml.
Quantitative detection of total HCV core antigen. Samples tested for quantification of HCV RNA were also assessed for total HCV CA by the Trak-C assay (Ortho-Clinical Diagnostics, Les Ulis, France). This novel assay is an ELISA-based method for microplate using 100 μl of serum performed according to the manufacturer's instructions. The range of detection is approximately 1.5 pg/ml to 300 pg/ml (about 4 to 6.4 log10 IU/ml).
HCV genotype determination. Patient samples were genotyped at D0 for all patients by using the Versant HCV genotype assay (Bayer Diagnostics) according to the manufacturer's instructions. This assay was done with 20 μl of amplicons from Cobas Amplicor technology.
Statistical analysis. The statistical analysis was performed with SPSS software version 10.1 for Windows (Statistical Package of Service Solutions; SPSS Inc., Chicago, Ill.). Results from CA quantification were corrected with a 104 factor and expressed in log10 (pg/ml · 104) for the correlation study with RNA quantification (log10 IU/ml). When quantification results (HCV RNA and CA) were under the cutoff value, we used the logarithm of half the cutoff value [2.49 log10 IU/ml for RNA and 3.88 log10 (pg/ml · 104) for CA]. The correlation study was done by Pearson's test. All statistical significance was assessed at the level where P was 0.05.
RESULTS
Study of the two markers at initiation of treatment. A very good correlation between HCV RNA and CA levels of the 348 patients at initiation of treatment (D0) was found (0.92, P < 0.01) (Fig. 1). This correlation at initiation of treatment was observed independently of type of response and HCV genotype (Table 1). Intra- and intergenotype sensitivity studies indicated no significant difference between HCV RNA and HCV CA for the four most frequent HCV genotypes (1, 2, 3, and 4) (Table 1 and Fig. 2).
Viral kinetics sorted by response to treatment. We obtained similar kinetics for the two viral markers, HCV RNA and HCV CA, for each type of per-protocol patient response when taking into account the different cutoff values of these assays (2.79 log10 RNA and 4.18 log10 CA) (Fig. 3). So, when the optical density is under the cutoff value, curves are not similar. Viral kinetics in nonresponders showed a mean transient decrease of about 1 log10 between D0 and W12 for both markers (1.07 ± 1.13 log10 RNA and 0.96 ± 0.86 log10 CA). In sustained responders, the early decrease (W4) of RNA and CA (3.12 ± 1.18 and 1.75 ± 1.02, respectively) was significantly different than that of relapsers (2.03 ± 1.22 and 1.68 ± 1.06, respectively; P < 0.001); the latter was also significantly different than that of nonresponders (0.83 ± 0.89 and 0.81 ± 0.74, respectively; P < 0.001). For patients presenting a breakthrough during treatment, decrease at W4 (2.49 ± 1.14 log10 RNA and 1.73 ± 0.86 log10 CA) was significantly different from that for RR and SVR (P < 0.01) (Table 2).
Predictive values of response and nonresponse. The negative predictive value (NPV) and the positive predictive value (PPV) of response to treatment were calculated according to HCV RNA or HCV CA below the cutoff value or to a decrease of HCV RNA or HCV CA superior than 2 log10 under treatment. NPV and PPV were studied at W4 and W12 (Table 3). The best PPV was observed using undetectable HCV RNA at W4 (72%). Finally, 71% of SVR had undetectable HCV RNA at W4. The best NPV was obtained at W12 using undetectable HCV CA alone as well as undetectable HCV RNA or a 2-log reduction for HCV RNA (99% for both parameters), allowing detection of 65% or 64% of NR, respectively.
DISCUSSION
In this study, we have evaluated for the first time the use of a new HCV marker (HCV CA) assay in comparison with a quantitative HCV RNA assay for baseline viral load and early virological response in HCV/HIV-coinfected patients included in the ANRS HC02 RIBAVIC study. It has been recommended from large pivotal trials (13, 16) to evaluate the early virological response after 12 weeks of treatment, based on a 2-log decline or a negative viral RNA algorithm (1, 14). This approach allows early discontinuation of treatment for patients who will not respond and, thus, could avoid side effects and expense for these patients. General application of this algorithm is dependent on HCV RNA quantitative methods, which are cost-effective and not accessible in every laboratory for patient follow-up.
Many studies have observed a very good correlation (0.72 < r < 0.80) between quantification methods of HCV RNA and HCV CA in HCV-monoinfected patients (7, 12, 20, 22, 24). In HCV/HIV-coinfected patients, we also found an excellent correlation at baseline between these two markers of HCV infection (0.92). In our study, 15 patients (4.4%) had an undetectable HCV CA at baseline, with detectable HCV RNA between 615 and 101,447 IU/ml (mean of 18,417 IU/ml). Soffredini et al. observed that 6% of HCV-monoinfected patients (7/111) had undetectable HCV CA with detectable RNA at initiation of IFN or pegylated IFN plus ribavirin treatment. The threshold of HCV CA quantification was also evaluated at 15,000 IU/ml (20). It is important to underline that only 1 patient among the 15 patients found to be CA negative and RNA positive at baseline was a nonresponder. Sensitivity of the HCV CA quantification may be increased with automated technology, such as using chemiluminescent detection with the Lumipulse HCV Ortho antigen assay (21). The range of HCV CA detection using Lumipulse is 1 to 1,000 pg/ml, whereas the Trak-C range is 1.5 to 100 pg/ml, and correlation with HCV RNA for Lumipulse is 0.87 (21). In our previous study of HCV RNA and CA correlation in HCV-monoinfected patients, we showed that 1 pg/ml of HCV CA was approximately equivalent to 10,000 IU/ml, as confirmed by Massaguer et al. (12). In HIV/HCV-coinfected patients, we observed a correspondence of 8,400 IU/pg at D0.
During IFN-ribavirin treatment, kinetics of the two HCV markers were similar (when optical density was above the cutoff value for both assays), whatever the arm of treatment, genotype, or HIV infection characteristics, as we observed with HCV-infected patients (24). Two early phases of viral elimination were observed with SVR, as previously described for HCV-infected, treated patients: a very early phase with a rapid viral decline, before W4, and a second phase with a slower decline, between W4 and W12 (15, 26). In RR, the first phase of decrease was slower than that of SVR and was prolonged up to W12. Indeed, it was shown that the rate of the phase 2 decay correlated closely with that of SVR to IFN-based treatment regimens (26). Especially for NR, a very weak decline was observed before W12. A viral rebound was always observed after W12, with return to baseline value. Unlike the results reported by Schüttler et al., our studies did not demonstrate any variations of HCV RNA and HCV CA ratio whatever the status of treatment (before or during treatment) (18). It was hypothesized that HCV RNA-free particles containing HCV CA were produced during treatment and secreted by infected cell or generated by in vivo degradation of HCV (18).
In the context of HIV/HCV-coinfected patients, early virological response is generally defined as an undetectable HCV RNA at W12 of treatment or as a 2 log10 decrease from baseline of HCV RNA level, as is defined for HCV-monoinfected patients (1, 5, 14). We have studied the PPV and NPV for several parameters, including undetectable HCV CA. We observed that the best NPV (99%) was obtained at W12 using three parameters individually, as described for HCV-monoinfected patients: undetectable HCV CA, significant decrease from D0 to W12 of HCV RNA, and significant decrease from D0 to W12 or undetectable HCV CA (10) (independently of genotype or arm of treatment). By contrast, a decrease of HCV CA of >2 log from D0 to W12 did not predict SVR because of the high threshold of this assay.
We have previously shown that the new HCV CA assay (Trak-C) is a valuable test, well correlated with the HCV RNA assay in the follow-up of patients with chronic hepatitis C treated with IFN plus ribavirin. This ELISA-based method is also cheaper (cost is 73% lower than that of quantitative PCR, as reported for Lumipulse) and easier to perform than molecular tests. However, its sensitivity needs to be improved, particularly in the assessment of the final response, as it is not sensitive enough to measure the sustained virological response (21). Chemiluminescence detection, as recently demonstrated, may increase the sensitivity of the assay as much as twofold (21). Moreover, the stability of HCV core antigen in clinical samples is higher than that of RNA (21).
In conclusion, we showed in this study that, for HCV/HIV coinfected patients, (i) Trak-C is well correlated with the HCV RNA assay whatever the HCV genotype and (ii) HCV CA could be an alternative to HCV RNA for therapeutic follow-up. Thus, the Trak-C core antigen assay could be useful as a low-priced alternative test to nucleic acid detection, particularly in developing countries.
ACKNOWLEDGMENTS
ANRS (French National Agency for Research on AIDS and Viral Hepatitis) was the sponsor of the ANRS HC02 RIBAVIC study, which was conducted with the support of Schering Plough (C. Lemonnier and A. Rimailho).
We thank all of the investigators and virologists involved in the ANRS HC02 RIBAVIC study in France (F. Bani-Sadr [Groupe Hospitalier Universitaire Est, Université Paris 6, Inserm U444], E. Rosenthal [H?pital de l'Archet, Faculté de médecine, Nice], A. Benzeckri and C. Degott [Groupe Hospitalier Universitaire Nord, Université Paris 7], C. Goujard [Groupe Hospitalier Universitaire Sud, Université Paris 11], G. Pialoux [Groupe Hospitalier Universitaire Est, Université Paris 6], L. Piroth [Centre Hospitalier Universitaire, Dijon], and D. Salmon-Céron [Groupe Hospitalier Universitaire Ouest, Université Paris 5]) and D. Lesteven from Ortho-Clinical Diagnostics, France, for supplying the Trak-C kits. Many thanks to Kevin L. Erwin, qualified biomedical translator, for proofreading the manuscript.
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Laboratoire de Bactériologie-Virologie-Hygiène Hospitalière, CHU Angers, France
1 Laboratoire de Virologie, CHU Grenoble, France
2 Inserm U707, Faculté de Médecine St Antoine, Paris, France
3 Service d’Hépatologie, H?pital Necker-Enfants Malades, Paris, France
4 Service de Médecine Interne, H?pital Pitié-Salpétrière, Paris, France
5 Service des Maladies Infectieuses et Tropicales, Hopital Raymond Poincaré, Paris, France(A. Pivert,C. Payan, P. Mo)