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Aptima 및 HCV 바이러스 Performance Evaluation of Aptima HBV and HCV Quant Assays in the Panther System

Background: Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain a serious health problem despite advancements in their pre-vention and treatment. Guidelines on their management recommend using viral DNA/RNA titers. Thus, accurate measurement and prompt report-ing are crucial. Methods: The performances of the Aptima HBV and HCV Quant assays (Hologic Inc., USA) were analyzed. The results were compared with those of Cobas 4800 (Roche Molecular Systems, USA). Linearity, limit of detection (LoD), and precision were evaluated as recommended in each corresponding CLSI guideline. Results: Passing-Bablok regression analysis showed a high correlation between the two assays: regression line was y = 0.0684+1.025x (95 % CI: 0.9604-1.092) for HBV and y = -0.9650+1.141x (95 % CI: 1.071-1.226) for HCV. Agreement between the assays’ qualitative results based on categorical analysis was 82.30 % (185/224) ( κ : 0.738, 95 % CI: 0.701-0.775) for HBV and 94.52 % (69/73) ( κ : 0.855, 95 % CI: 0.796-0.914) for HCV. The LoD values for HBV and HCV were 4.448 IU/mL and 6.166 IU/mL, respectively. The percent coefficient of variation ( % CV) for the HBV assay for both low and high positive controls was less than 2 % , whereas for the HCV assay, % CV for low positive control was 3.20 % . Conclusions: Overall, the Aptima HBV and HCV Quant assays demonstrated a high correlation with Cobas 4800. These tests were both sensitive and precise. Therefore, we conclude that the Aptima assay is a practical tool in the management of HBV- and HCV-infected patients.


INTRODUCTION
Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain a serious health problem despite the existence of an effective vaccine and antiviral drugs. The prevalence of HBV infection line, HCV RNA titer measurement is needed to make a diagnosis, monitor treatment ef cacy, and test for reinfection [6]. Thus, accurate measurement of HBV DNA and HCV RNA titers is crucial for the proper management of HBV and HCV infections.
Instruments measuring viral genetic material mostly use a realtime, quantitative polymerase chain reaction (PCR) method whereas the Aptima assay (Hologic Inc., San Diego, CA, USA) uses transcription-mediated ampli cation (TMA) [7]. In addition, the platform that the Aptima assay utilizes, the Panther system, offers a fully-automated random access system, allowing prompter results and less hands-on time for clinical pathologists carrying out the task [8].
In this paper, Aptima HBV and HCV assays' linearity, limit of detection (LoD), precision, and correlation with Cobas 4800 (Roche Molecular Systems, Pleasanton, CA, USA) were evaluated to assess the analytical performance of these assays.

Clinical specimens
For the comparison study, residual plasma samples that had been tested with Cobas 4800 for either HBV or HCV genetic material were used; 224 HBV residual samples and 73 HCV residual samples were collected. They had been stored at -80°C for 9 months, at most.

Aptima HBV and HCV Quant assays
The viral genetic load was measured using Aptima HBV and HCV Quant assays. The Aptima HBV Quant assay uses real-time TMA technology to quantify HBV DNA. It targets two highly-conserved regions in the virus's polymerase and surface genes to increase the assay's tolerance for potential mutations. These targets are captured using capture oligonucleotides coupled to magnetic beads, which can be separated from the specimen in a magnetic eld. Once the target is ampli ed, uorescently-labeled probes (torches) are used for detection. The time taken for the uorescent signal to reach a speci ed threshold is inversely proportional to the starting HBV concentration. The concentration of a sample is determined automatically by the Panther system software. The procedure for the HCV assay is almost identical to that of HBV.
The Aptima HCV assay targets two highly-conserved regions at the relatively large 5'UTR of the HCV genome.

Precision, Linearity, and LoD
Precision was assessed using 3 different target concentrations or 3 control samples (negative, low positive, and high positive controls) that were included in each assay. Linearity was con rmed using 7 concentrations provided in the AcroMetrix HBV, HCV Panel 1.2 mL (Thermo sher Scienti c) [9]. Among these, the ones with the lowest HBV DNA and HCV RNA concentrations (50 IU/mL) were used for serial dilution to estimate the LoD. Five concentrations were made and measured 20 times each using the Aptima assay [10].

1) HBV
Of the 224 samples collected, only 84 were quanti ed in both assays. The results were compared using Passing-Bablok regression and Bland-Altman analysis. Passing-Bablok regression analysis demonstrated a high correlation between the two data sets, with a regression line slope of 1.025 (95% CI: 0.9604-1.092) and a correlation coef cient r of 0.979 (Fig. 1). Average bias between them was -0.19 Log IU/mL (SD = 0.34 IU/mL) (Cobas 4800-Aptima), with no speci c trend as the HBV titer increased. Aptima quanti ed more than two standard deviations higher than Cobas 4800 for two samples, and more than three standard deviations higher for one sample (Fig. 2). Categorical distribution of each sample is summarized in Table 1 For four samples, Aptima quanti ed more than two standard deviations lower than Cobas 4800 (Fig. 4)

Linearity
To validate the linearity of both Aptima HBV and HCV assays, 2 replicates of 7 points from the AcroMetrix HBV, HCV Panel 1.  Abbreviations: LLOQ, lower limit of quantification; Pos. < LLOQ, Detected but below the lower limit of quantification.

LoD
To estimate the LoD, serial dilution using a sample from the Ac-

Precision
To evaluate the precision of the Aptima HBV and HCV assay, the percent coef cient of variation (%CV) was calculated by measuring the concentration of 3 control samples (negative, low positive, and high positive controls) twice each day for 10 days.

1) HBV
Runs using negative control always resulted in no detection, so %CV could not be calculated. The %CV for low positive control (2.88 Log IU/mL; SD = 0.05) was 1.66%, and for high positive control (4.52 Log IU/mL; SD = 0.04), it was 0.95%.

2) HCV
Runs using negative control always resulted in no detection, so %CV could not be calculated. The %CV for low positive control (2.26 Log IU/mL; SD = 0.07) was 3.20%, and for high positive control (5.44 Log IU/mL; SD = 0.09), it was 1.69%.

DISCUSSION
In this study, we evaluated the analytical performances of the  [11]. Their average difference (Aptima-CA PCTMv2) was -0.195 Log IU/mL [11]. In another article comparing Aptima HCV assay with the COBAS Ampliprep/CO-BAS TaqMan HCV RNA Test v2.0, the average difference (Aptima-Abbott) was 0.11, which was similar to the average difference obtained in our study [12]. According to the categorical distribution of each sample shown in Table 1, 27 out of the 86 HBV samples that Cobas 4800 reported as "Not detected" were positive but below the LLOQ according to the Aptima assay. In contrast, only 6 of the 65 samples that Aptima reported as "Not detected" were reported as positive but below the LLOQ by Cobas    4800. The four samples that were below the LLOQ in the Aptima assay but quanti ed in Cobas 4800 had very low concentrations, ranging from 11.1 IU/mL to 25.4 IU/mL. The two samples that were below the LLOQ in the Cobas 4800 assay but quanti ed in Aptima had very low concentrations as well. Their exact numbers were 10.0 IU/mL and 12.6 IU/mL. The qualitative results of the two assays showed substantial agreement, with a Cohen's coef cient κ of 0.738 for HBV and 0.855 for HCV. The Aptima HBV assay quanti ed more than two standard deviations higher than Cobas 4800 for two samples and more than three standard deviations higher for one sample (Fig. 2). In the case of HCV, Aptima quanti ed more than two standard deviations lower than Cobas 4800 for four samples (Fig. 4). Prior studies have demonstrated that such discrepancies were statistically related to a certain genotype of the virus, although its clinical signi cance was less clear [11]. Genotyping of the discrepant samples in our study could not be done due to a lack of remnant samples.
The LoD values for HBV and HCV were 4.448 IU/mL and 6.166 IU/mL, respectively. In the case of HBV, the estimated value was in accordance with the LoD stated on the package insert paper [12,13]. In contrast, the LoD of the HCV assay differed from that stated on the package insert paper. It was 3.9 IU/mL for plasma [14], which was even lower than the lower LoD range of 95% CI obtained in our study. However, when compared to the LoD of the Cobas 4800 HCV assay, which was 9.2 IU/mL (95% CI: 7.8-11.5 IU/ mL), the LoD obtained in our study was lower, albeit with an overlapping 95% CI [15].
The Aptima HBV and HCV assays were both found to be precise. The %CV of the HBV assay for low positive control was 1.66%, and for high positive control, it was 0.95%. While these were both less than 2%, the %CV of HCV for low positive control was 3.20%, and for high positive control, it was 1.69%. In spite of the fact that the %CV of HCV for low positive control exceeded 3%, this value was similar to what prior studies obtained at low concentrations with the Aptima HCV assay [12], and it is better when compared to studies that used other HCV assays [16].
Even though the analytical performance of the Aptima assays was evaluated as satisfactory, our study had some limitations. Regarding samples that showed a discrepancy between Cobas 4800 and Aptima assays, genotyping of the viruses could not be done due to a lack of remnant samples. In addition, the number of HCV samples was too few, and the samples were mostly high-titered. If we had collected more positive samples in the lower range, we might have gotten better results. As for the difference between the measured LoD and the LoD stated in the insert paper for HCV, if we had measured each concentration more, our estimation might have been closer to the stated LoD, and the 95% CI would have been narrower as well.
Despite these limitations, we found the results of the Aptima assay to be highly correlated to that of Cobas 4800. Moreover, according to the other parameters that we evaluated, Aptima was both sensitive and precise. Therefore, we conclude that the Aptima assay is a practical tool in the diagnosis and management of HBV-and HCV-infected patients.

Conflicts of Interest
None declared.