Evaluation of the Core Antigen Assay as a Second-Line Supplemental Test for Diagnosis of Active Hepatitis C Virus Infection

MATERIALS AND METHODS

RESULTS

The percentage of individuals actively infected with HCV, as demonstrated by detection of HCV core antigen and HCV RNA in anti-HCV antibody-positive clinical specimens from the treatment-naïve population evaluated in the present study, is shown in Fig. 1. Of note, 110 of 478 (23%) of the specimens showed no evidence of active infection by any of the three assays, while 368 of 478 (77%) of specimens showed evidence of active infection by one or more of the assays. The prevalence of active infection among the seropositive individuals in our study population was similar to that observed in other studies (1, 18, 34). HCV core antigen was detected by OTCA in 331 of 478 (69%) specimens, and HCV RNA was detected by the quantitative HCV bDNA assay in 348 of 478 (73%) specimens. Testing by the more sensitive qualitative HCV TMA detected HCV RNA in an additional 19 specimens, of which 2 were reactive for HCV core antigen by OTCA and 17 were not reactive for HCV core antigen by OTCA. This yielded an overall rate of HCV RNA detection of 76.8% (367 of 478 specimens). The reported quantities of HCV core antigen in the two specimens that had detectable HCV RNA by HCV TMA but in which the viral load was below the limit of quantification of the HCV bDNA assay were 268 and 3.3 pg/ml, respectively. In addition, a third specimen was reactive for HCV core antigen with a quantity of 1.7 pg/ml, just above the 1.5-pg/ml assay cutoff, but was not reactive for HCV RNA by either the HCV bDNA assay or HCV TMA. These results highlight the excellent specificity of OTCA, since only 1 of 331 (0.3%) anti-HCV-positive specimens that were reactive for HCV core antigen had no evidence of HCV RNA. However, OTCA showed a lower sensitivity, since HCV core antigen was not detected in 37 of 367 (10%) anti-HCV-positive specimens from treatment-naïve individuals who had active HCV infection, as documented by testing for HCV RNA. Therefore, the detection of active infection would have been missed in 37 of 478 (7.7%) of specimens from this population if the present OTCA had been used as a second-line confirmatory test.

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FIG. 1.

Percentage of active infections demonstrated by detection of HCV core antigen by OTCA versus detection of HCV RNA by the quantitative HCV bDNA assay and/or the qualitative HCV TMA.

The viral load distribution and HCV core antigen status of the 348 specimens that had quantifiable HCV RNA by the quantitative HCV bDNA assay are shown in Fig. 2. The HCV RNA concentrations of the 348 specimens ranged from 2.8 to 6.8 log₁₀ IU/ml. HCV core antigen was not detected in 20 specimens that had HCV RNA concentrations between 2.8 and 4.8 log₁₀ IU/ml (704 and 55,000 IU/ml), of which 5 specimens that had sufficient volume for retesting were reactive for HCV RNA by the qualitative AMPLICOR HCV (version 2.0) assay. In addition, HCV core antigen was not detected in 17 specimens that were reactive for HCV RNA by HCV TMA but in which the virus was present at levels below the quantification limit of the HCV bDNA assay (and, therefore, whose data are not shown on the histogram). Eleven of these 17 specimens had sufficient volume for retesting by the qualitative AMPLICOR HCV (version 2.0) assay, of which 7 were reactive for HCV RNA, 1 was indeterminate for HCV RNA by two successive tests, and 3 were not reactive for HCV RNA. Of the three specimens that were not reactive, two with sufficient residual volume were found to be negative for HCV RNA upon repeat testing by HCV TMA. Taken together, these results show that HCV core antigen was not reliably detected in specimens from patients with active infection when HCV RNA levels were 4.8 log₁₀ IU/ml or less.

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FIG. 2.

Viral load distribution and HCV core antigen detection status of clinical specimens confirmed to be positive for anti-HCV antibodies.

The correlation between the HCV core antigen concentration and the HCV RNA load for 328 specimens in which active infection was detected by both OTCA and the HCV bDNA assay is shown in Fig. 3. For this analysis, 72 of 328 (22%) of the specimens tested had absorbance values above the linear range of OTCA and thus required dilution and retesting. By comparison, only 1 of 328 (0.3%) of the specimens required dilution and retesting by the HCV bDNA assay. Deming regression analysis of these 328 specimens yielded the equation y = 0.98x − 3.59 with a correlation coefficient of 0.915, where y is the concentration of HCV core antigen in log₁₀ picograms per milliliter and x is the concentration of HCV RNA in log₁₀ international units per milliliter. This analysis indicated that the relationship between the concentrations of HCV core antigen and HCV RNA was consistent throughout the common dynamic ranges of the assays. The difference in mean values between the HCV core antigen concentration (in picograms per milliliter) and the HCV RNA concentration (in international units per milliliter) was determined to be 3.72 log₁₀. On the basis of the results of this analysis, 1 pg of HCV core antigen per ml corresponds to 5,200 IU (95% confidence interval [CI], 4,800 to 5,600 IU/ml) of HCV RNA per ml.

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FIG. 3.

Correlation between the concentrations of HCV core antigen measured by OTCA and HCV RNA measured by the HCV bDNA assay. The results of both assays for 328 clinical specimens were analyzed by Deming regression analysis.

Subsequently, HCV genotype determination was requested for 179 of the 348 patients shown to be viremic. The distribution of genotypes for these patients was as follows: 108 (60%) genotype 1, 20 (11%) genotype 2, 44 (25%) genotype 3, 3 (1.5%) genotype 4, 3 (1.5%) genotype 6, and 1 (0.5%) mixed genotypes. This distribution is commensurate with that documented by BCCDC for HCV-infected patients in British Columbia over the past 10 years of 65% genotype 1, 12% genotype 2, 22% genotype 3, and 2% for genotypes 4, 5, and 6 (BCCDC, unpublished data). As before, Deming regression analysis and calculation of the differences in mean values between the HCV core antigen concentration (in picograms per milliliter) and the HCV RNA concentration (in international units per milliliter) was performed for patients who were infected with HCV genotype 1, 2, or 3 and who had active infection, as detected by both OTCA and the HCV bDNA assay. This analysis indicated that there are consistent relationships between HCV core antigen concentration results and HCV RNA concentration results. On the basis of the mean log₁₀ differences in results between the assays, 1 pg of HCV core antigen per ml corresponds to HCV RNA concentrations of 5,044 IU/ml (95% CI, 4,995 to 5,094 IU/ml) for genotype 1, 4,845 IU/ml (95% CI, 4,372 to 5,371 IU/ml) for genotype 2, and 6,835 IU/ml (95% CI, 6,566 to 7,116 IU/ml) for genotype 3.

The LoD of OTCA was estimated by testing a dilution panel of the WHO HCV standard (Fig. 4). The results showed that OTCA detected core antigen in 5 of 24, 20 of 20, 19 of 20, and 12 of 12 replicates of dilutions with estimated HCV RNA concentrations of 10,000, 20,000, 50,000, and 75,000 IU/ml, respectively. No core antigen was detected in dilutions containing 5,000 IU of HCV RNA per ml or less. These data were analyzed by linear regression analysis to determine the LoD, or the analyte detection sensitivity, which is defined as the minimum analyte concentration at which the assay could distinguish a reactive specimen from a nonreactive specimen 95% of the time. This definition of LoD has been recommended in the National Committee for Clinical Laboratory Standards protocol for qualitative molecular methods (26). The x coordinate where the calculated curve intercepts the 95% detection line indicates that the estimated LoD of OTCA is 27,000 IU/ml (95% CI, 20,000 to 49,000 IU/ml).

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FIG. 4.

LoD of OTCA estimated by application of a linear logistic regression model to the results obtained with the WHO dilution panel. The dashed lines represent the upper and lower 95% confidence levels (CL). The estimated LoD of 27,000 IU of HCV RNA/ml is indicated by the x coordinate, where the calculated curve intersects the 95% detection line.

DISCUSSION

Proper clinical diagnosis and management of HCV-infected individuals require both serological screening for anti-HCV antibodies and qualitative HCV RNA testing (27). Despite the declining incidence of HCV infections in North America over the past 15 years, the prevalence of HCV infections worldwide continues to pose a significant disease burden; more patients are expected to be diagnosed with the disease in the next two decades because of the long latent period prior to the first clinical signs of liver impairment (18). Hence, diagnostic laboratories will likely face increasingly high demands for accurate diagnostic screening and confirmation of active HCV infection.

As the largest reference laboratory in Canada on the basis of the population served, BCCDC performs approximately 95% of the HCV antibody tests for the 4 million inhabitants of the province of British Columbia. In the past year, BCCDC performed 96,000 anti-HCV antibody tests, of which approximately 4,800 (5%) showed some degree of reactivity and therefore required a second test for confirmation of active HCV infection. Although qualitative HCV RNA testing by an assay with a sensitivity of 50 IU/ml is the laboratory's method of choice at present, given the large volume of specimens tested annually, we wished to explore simpler technologies that could be used to confirm active HCV infection. As a potential alternative to HCV RNA testing, we evaluated OTCA as a second-line confirmatory test for the diagnosis of active HCV infection. OTCA offers a number of practical advantages: (i) it can be performed in less time than HCV RNA tests; (ii) it does not require specific training in molecular techniques; and (iii) as an EIA, it is anticipated that OTCA might be less expensive than HCV RNA tests. Also, OTCA is theoretically not as susceptible to carryover contamination as HCV RNA tests and can therefore be performed with the same sample used for the initial anti-HCV antibody test. However, a second aliquot would still be required to minimize the risk of HCV RNA carryover if core antigen-negative specimens are to be reflexed for additional qualitative HCV RNA testing.

It is important to evaluate an assay in the context of its intended use, since an assay may provide great utility in one setting but less utility in another. To our knowledge, ours is the first study to evaluate OTCA as a supplemental test in the diagnostic setting once seroconversion has been confirmed. Previous studies have evaluated the utility of OTCA for the screening of blood products, focusing on its potential to shorten the time to the identification of active infection prior to antibody production during the window period (12, 16, 17). Other studies have examined the assay's utility in the monitoring of therapy, as assessed by its ability to assess the presence or the absence HCV core antigen at a given time point or to examine the kinetics of HCV core antigen decline (22, 32, 36, 37). Many of the previously published studies reported on the performance of an earlier prototype version of OTCA, which purportedly is less sensitive than the present version evaluated here. Although a number of the earlier studies have estimated the sensitivity of the assay, none have defined the LoD by assessing multiple dilutions and multiple replicates of a WHO specimen panel.

Our findings show that OTCA was unable to confirm active HCV infection in a significant proportion of specimens. No core antigen was detected in 30.8% of specimens tested for confirmation of active infection, of which 25.2% were shown to be viremic by HCV RNA testing. Hence, if OTCA were used as a second-line test by using the current cutoff recommended by the manufacturer, at least 30.8% of specimens would have required testing for HCV RNA to confirm active HCV infection. Although HCV core antigen was detected in all samples in our study with viral loads of 4.8 log₁₀ HCV RNA IU/ml or greater, the results of our study suggest that HCV core antigen may not be detected by the present OTCA in specimens with HCV RNA levels below 4.8 log₁₀ IU/ml (55,000 IU/ml). These findings are consistent with those of other investigators who reported on the sensitivity of OTCA. For example, Icardi et al. (12) showed that core antigen was not detected in 6 of 103 (5.8%) HCV RNA-positive, anti-HCV-negative specimens that were sent for blood screening, while Zanetti et al. (37) showed that core antigen was undetectable in 21 of 82 (26%) of HCV RNA-positive specimens collected from patients before, during, and after therapy for HCV infection. Similar to our findings, the viremic specimens in which core antigen was not detected by OTCA had HCV RNA levels up to 48,600 IU/ml.

Our correlations between the concentrations of HCV core antigen (in picograms per milliliter) and the concentrations of HCV RNA (in international units per milliliter) are also consistent with those reported in earlier studies. Bouvier-Alias et al. (6) reported correlations of 1 pg of HCV core antigen per ml to 7,688 IU of HCV RNA per ml for genotype 1b and to 6,711 IU of HCV RNA per ml for genotype 3. Similarly, the correlations estimated from the differences between the mean concentrations of HCV core antigen and HCV RNA for each genotype reported by Veillon et al. (36) suggest that 1 pg of HCV core antigen per ml corresponds to HCV RNA concentrations of 8,128 IU/ml for genotype 1, 8,511 IU/ml for genotype 2, and 9,550 IU/ml for genotype 3. The variation among the reported correlations between HCV core antigen and HCV RNA concentrations may be explained in part by the use of different quantitative assays and specimens from different patient populations in the various studies. While our study used the third-generation HCV bDNA assay, Veillon et al. (36) used the second-generation version of this assay and the AMPLICOR HCV MONITOR test (version 2.0; Roche Molecular Diagnostics), and Bouvier-Alias et al. (6) used the third-generation HCV bDNA assay as well as the AMPLICOR HCV MONITOR test (version 2.0) and the SuperQuant assay (National Genetics Institute, Culver City, Calif.). Even though the quantification results were expressed in the same international units, the quantification results reported from the different HCV RNA assays may not be equivalent (25). Also, while our study population consisted exclusively of treatment-naïve patients, the studies of Veillon et al. (36) and Bouvier-Alias et al. (6) included patients undergoing therapy. Not only do the mean values for HCV core antigen and HCV RNA levels differ between these patient groups, but the relationship between HCV core antigen and HCV RNA concentrations may also vary between patients (6, 16). For all of these reasons, the reported correlations between the concentration of HCV core antigen and the HCV RNA concentration may vary from study to study.

The relatively poor sensitivity of OTCA compared to those of nucleic acid-based tests accounts in part for its inability to detect core antigen in some HCV RNA-positive specimens. Other investigators have noted a comparatively poor sensitivity of OTCA in its present and earlier formats. In evaluating a prototype version of OTCA for which the manufacturer set a less sensitive cutoff of 2.0 pg/ml, Bouvier-Alias et al. (6) found that the assay consistently detected core antigen in Acrometrix panel members containing ≥200,000 IU of HCV RNA per ml but in only one of nine replicates of a panel member containing 50,000 IU of HCV RNA per ml. Veillon et al. (36) evaluated the present version of OTCA, for which the manufacturer has set a lower detection limit of 1.5 pg/ml, by testing dilutions of the WHO standard and found that the assay detected core antigen in three of three replicates of each of the 1:4, 1:2, and 1:1 dilutions containing estimated HCV RNA concentrations of ∼12,500, ∼25,000, and ∼50,000 IU/ml, respectively. Our study also tested dilutions of the WHO standard and determined a LoD for OTCA of 27,000 IU/ml (95% CI, 20,000 to 49,000 IU/ml), which represents the viral level at which OTCA detects core antigen in HCV RNA-positive specimens 95% of the time. The LoD should not be confused with the manufacturer's cutoff, which is not necessarily based on the 95% detection limit. The LoD of 27,000 IU/ml that we determined is consistent with our findings of the ability of OTCA to detect core antigen in clinical specimens. Most viremic specimens (36 of 37 [97%]) in which core antigen was not detected had HCV RNA levels below 27,000 IU/ml, although 1 specimen that was not reactive for core antigen had a viral level of 55,000 IU of HCV RNA/ml.

At its present level of sensitivity, OTCA has limited value as a second-line confirmatory test for the diagnosis of active HCV infection because a significant percentage of viremic individuals have viral loads below its detection limit. OTCA did not detect core antigen in 10% (37 of 367) of viremic samples or in 7.7% (37 of 478) of all samples from the entire population. Given that nucleic acid-based tests are 2 to 4 orders of magnitude more sensitive than OTCA, they are better able to detect viremia. For example, the limit of quantification of the HCV bDNA assay is 615 IU/ml, and the LoD of the qualitative HCV TMA is ≤9.6 IU/ml. Similarly, the limit of quantification of the quantitative AMPLICOR HCV MONITOR (version 2.0) test is 600 IU/ml (20) and the LoD of the qualitative AMPLICOR HCV (version 2.0) test is 50 to 100 IU/ml (15; AMPLICOR HCV test, version 2.0; Roche Molecular Systems, 2001 [http://www.fda.gov/cdrh/pdf/p000010.html ]). Therefore, at present qualitative nucleic acid-based testing remains the standard for the confirmation of active HCV infection in the diagnostic setting.