Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major causes of chronic liver disease. HBV infection affects nearly 250 million individuals worldwide and the number of individuals chronically infected with the HCV is estimated to range between 130 and 150 million [1,2]. Chronic HBV infection may lead to cirrhosis, decompensated liver failure, and hepatocellular carcinoma (HCC), all of which carry significant morbidity and mortality [3,4]. Similarly, in HCV infections, although most chronically infected individuals are asymptomatic, 20%–30% develop cirrhosis over a period of 20–30 years, and 1%–4% of patients with cirrhosis develop HCC annually [5]. Despite the availability of potent antiviral treatments, HCV and HBV infections remain major health concerns worldwide.

Current HBV treatment is based on the lifelong administration of nucleoside or nucleotide analogs. These medications maintain undetectable HBV DNA levels in the long term, substantially improve the prognosis of HBV-related liver disease, and reduce HCC incidence [6,7]. Current therapeutic research focuses on developing strategies that lead to hepatitis B surface antigen (HBsAg) clearance and possible anti-HBsAg seroconversion, which represents a functional cure for HBV infection. The goal of HCV therapy is to cure the infection as judged by a sustained virological response, which is defined as an undetectable HCV RNA plasma/serum concentration at 12 or 24 weeks after treatment completion using a sensitive HCV RNA quantitation assay with a limit of quantitation of <25 IU/mL [8]. Therefore, HCV RNA should be monitored using sensitive real-time quantitative assays.

Human immunodeficiency virus type 1 (HIV-1) infection is a significant global health challenge. According to HIV treatment guidelines, antiretroviral therapy (ART) is effective when it leads to undetectable HIV-1 RNA in the plasma. However, results above 50 copies/mL may require further investigation [9]. Since this threshold is close to the lower limit of quantitation for most commercially available assays (20–75 copies/mL), assay performance at low HIV-1 RNA levels is important for decision-making during ART.

Accordingly, sensitive and accurate detection and quantification of viral load are essential for diagnosing HBV, HCV, and HIV-1 infections, establishing the prognosis of virus-related diseases, guiding the treatment decisions, and monitoring the response to antiviral treatment and the emergence of resistance. The Hologic Aptima Quant assay (Hologic Inc., Marlborough, MA, USA) is a fully integrated and automated quantitative assay, which is based on real-time transcription-mediated amplification technology using the Panthers system. In the present study, we evaluated the performance of the Hologic Aptima Quant assay for HBV, HCV, and HIV-1 for measuring viral load and compared the results with those obtained with the Abbott Alinity m system (Abbott Laboratories, Abbott Park, IL, USA).

1. Sample collection

This study was approved by the Research Ethics Committee of Dong-A University Hospital (DAUHIRB-20-127), and the requirement for informed consent was waived. Residual samples from HBV-, HCV-, or HIV-1 positive patients referred to the laboratory were used for comparison. Commercial controls were also included to ensure precision and linearity.

2. Precision

We measured the negative, low positive, and high positive controls using Hologic Aptima Quant assay twice daily for five consecutive days, following the guidelines established by Clinical and Laboratory Standards Institute (CLSI) [10].

3. Linearity

Linearity was evaluated using AcroMetrix HBV (50–50,000,000 log IU/mL), HCV (100–25,000,000 log IU/mL), and HIV-1 (100–5,000,000 copies/mL) panels from Thermo Fisher Scientific. Each dilution was run in duplicate according to CLSI guidelines [11]. Panels ranged in concentration from 7.52 to 1.39 log IU/mL for HBV, from 7.54 to 1.87 log IU/mL for HCV, and from 6.66 to 2.33 log IU/mL HIV-1. The results obtained from Hologic Aptima Quant assay for HBV was further evaluated by diluting HBV-positive clinical specimens with HBV-negative serum. Linear regression analysis was performed, and the concordance correlation coefficient (R²) was calculated.

4. Limit of detection

To verify the limit of detection, we tested patient samples at least 10 times (20 times for HBV and 10 times for HCV and HIV-1) using patient samples with lower limits of detection for each viral concentration. The patient samples ranged from 5 to 25 IU/mL, 5 to 25 IU/mL, and 20 to 40 copies/mL for HBV, HCV, and HIV-1, respectively.

5. Cross-reactivity

The specificity of the HBV assay was evaluated by analyzing 10 samples that tested negative by the Abbott Alinity m assay but positive for HCV, HIV-1, or cytomegalovirus. The aim was to assess the potential cross-reactivity with these viruses. We repeated the same procedures to evaluate the specificity of the HCV and HIV-1 assays.

6. Interference

Interference was evaluated using high concentrations of hemoglobin, bilirubin, and triglycerides. Five patient serum samples exhibiting visible signs of hemolysis, icterus, or lipemia were obtained. The presence of these interferences was confirmed using the ARCHITECT analyzer (Abbott Diagnostics, Abbott Park, IL, USA) and HIL (hemolysis, icterus, and lipemia) indices.

7. Method comparison

To compare the performance of Alinity m system with Hologic Panther Aptima system, a dataset comprising of 111 HBV-positive, 46 HCV-positive, and 50 HIV-1-positive samples with a wide range of quantitation values was compiled. The R² value was determined using Deming-regression analysis for each virus (HBV, HCV, and HIV-1). Additionally, a difference plot (Bland-Altman analysis) was also used. MedCalc ver. 19.0 (MedCalc, Ostend, Belgium) was used for statistical analysis, and a P-value <0.05 was considered significant.

The results of the precision study indicate that the coefficient of variation (CV) of precision was less than 5% for all viral targets, indicating a high level of consistency in the assay performance. Specifically, the CV of precision were 2.5%, 2.9%, and 2.8% (low control) and 0.7%, 3.1%, and 0.6% (high control) for the HBV, HCV, and HIV-1 assays, respectively. The linearity of viral load measurement was excellent for the assays (R²>0.99, for HBV, HCV, and HIV-1) (Fig. 1). The linearity of the Hologic Aptima Quant assay for HBV, as measured by diluting HBV-positive clinical specimens with HBV-negative serum, was also excellent (Fig. 2). The limit of detection for the assay was 10 IU/mL for both HBV and HCV, and 20 copies/mL for HIV-1, with 100% replicates detected. All samples gave a “not detected” result, indicating a cross-reactivity of 0%. High concentrations of hemoglobin, bilirubin, or triglycerides did not affect the performance of HBV, HCV, or HIV-1 assays.

Figure 1. Linearity for the Hologic Aptima Quant assay. (A) HBV. (B) HCV. (C) HIV-1. To evaluate the linearity, each member of the AcroMetrix panel was tested 2 times in the same experiment with the Hologic Aptima Quant assay, and the measured value were compared to the expected ones. The instruments were from the following company: Hologic Aptima Quant assay (Hologic Inc., Marlborough, MA, USA). Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus type 1.

Figure 2. Linearity for the Hologic Aptima HBV Quant assay. The instruments were from the following company: Hologic Aptima Quant assay (Hologic Inc., Marlborough, MA, USA). Abbreviation: HBV, hepatitis B virus.

When comparing the results of 111 of HBV-, 46 of HCV-, and 50 HIV-1-infected samples using both systems, viral load results were strongly correlated (R²=0.94–0.97). Strong correlations for HBV (R²=0.95), HCV (R²=0.94), and HIV-1 (R²=0.97) viral load were observed between the results obtained from the Abbott Alinity m and Hologic Aptima Quant assays. The values of the slope, intercept, and R from the Hologic Aptima Quant assay over the Abbott Alinity m assay represented by Bland-Altman analysis was 0.99, 0.13, and 0.98 for HBV; 0.99, 0.07, and 0.97 for HCV; and 1.05, 0.16, and 0.97 for HIV-1, respectively. Bland-Altman analysis showed that the difference between the mean results of the assays was small (0.16 log IU/mL for HBV, 0.01 log IU/mL for HCV, and 0.32 log IU/mL for HIV-1) (Fig. 3).

Figure 3. Deming correlation and Bland-Altman plot analysis of viral load results measured with the Hologic Aptima Quant assay compared with those measured by the Abbott Alinity m assay. (A) Deming regression of HBV DNA levels measured by the Hologic Aptima Quant assay and the Abbott Alinity m assay. (B) Bland-Altman plot analysis of the Hologic Aptima Quant assay and the Abbott Alinity m assay. (C) Deming regression analysis of HCV RNA levels measured by the Hologic Aptima Quant assay versus the Abbott Alinity m assay. (D) Bland-Altman plot analysis of the the Hologic Aptima Quant assay versus the Abbott Alinity m assay. (E) Deming regression analysis of HIV RNA levels measured by the Hologic Aptima Quant assay versus the Abbott Alinity m assay. (F) Bland-Altman plot analysis of the the Hologic Aptima Quant assay versus the Abbott Alinity m assay. In the Bland-Altman plots, the dotted and dashed lines represent mean differences 2SD, respectively. The instruments were from the following companies: Hologic Aptima Quant assay (Hologic Inc., Marlborough, MA, USA) and Abbott Alinity m system (Abbott Laboratories, Abbott Park, IL, USA). Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus type 1; SD, standard deviation.

Sensitive and accurate nucleic acid amplification technologies are recommended by International Clinical Practice Guidelines for the quantification of viral load in clinical practice, encompassing viruses such as HBV, HCV, HIV, and others. In the present study, we conducted the first comparison of the performance of the Hologic Aptima Quant assay with that of the Abbott Alinity m assay. Our evaluation demonstrates that the Hologic Aptima Quant assay meets the performance requirements necessary for clinical diagnosis. Previous studies utilizing single-item Hologic Aptima Quant assays have also yielded excellent results. For instance, a study comparing the Aptima HBV Quant assay with two commercial real-time polymerase chain reaction comparators revealed that the Aptima HBV Quant assay exhibited sensitivity, specificity, reproducibility, and accurately quantification of HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including those patients receiving antiviral treatment with nucleoside or nucleotide analogs [12]. Similarly, a recent study has compared the performance of the Hologic Aptima HCV Quant assay to that of the Roche Cobas TaqMan HCV test ver. 2.0, using the High Pure system (HPS/CTM), and found that the Hologic Aptima HCV Quant assay showed performance characteristics comparable to those of HPS/CTM, with increased sensitivity [13]. Sahoo et al. [14] conducted a study comparing the clinical performance of the Aptima HIV-1 Quant Dx assay with that of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test ver. 2.0 (CAP/CTM). Their findings demonstrated that the Aptima HIV-1 Quant Dx assay provided a suitable alternative for HIV-1 load monitoring [14].

To be effective as a diagnostic and viral load monitoring test, a nucleic acid amplification test assay must quantitate viral load across a wide dynamic range and demonstrate sensitivity, specificity, accuracy, and precision. The linearity of the Hologic Aptima Quant assay across a wide dynamic range is shown in Figs. 1 and 2. The R² values for samples diluted over an 8-log range of HBV concentrations was determined to be 0.998. Modest differences in the Hologic Aptima Quant assay results were observed for all members of the standard panel, a finding further confirmed in clinical specimens when compared to the Abbott Alinity m assay. The intercepts for the linear plots were calculated to be 0.13 log IU/mL for HBV, 0.07 log IU/mL for HCV, and 0.16 log copies/mL for HIV-1, indicating minimal bias and good linearity. With sensitivities of 10 IU/mL for HBV and HCV assay, and 20 copies/mL for HIV-1 assay, the Hologic Aptima Quant assay can accurately quantify viral loads at the levels necessary for effective treatment monitoring and diagnostics.

Cross-reactivity is also important to avoid false-positive results when diagnosing viral infections. Cross-reactivity of the Hologic Aptima Quant assay was 0%. Precision is indeed crucial for patient monitoring during treatment. The results of the precision study revealed that the CV of precision ranged from 1.3% to 3.92% for the HBV assay, 0.5% to 2.68% for the HCV assay, and 0.62% to 2.05% for the HIV-1 assay. Such low variation underscores the high level of confidence in the Hologic Aptima Quant assay to deliver reliable results from a single DNA/RNA measurement.

The high sensitivity of Hologic Aptima Quant assay, combined with the complete automation and high throughput provided by the Panther system, renders it an appealing option for any clinical laboratory. Moreover, fully automated systems reduce the demand for skilled personnel and minimize the possibility of human errors. Additionally, the use of a single test helps to reduce the additional costs associated with conducting separate tests to confirm infection.

In conclusion, the present study is the first to highlight the excellent sensitivity and precision of the newly developed Hologic Aptima Quant assay. This assay can be used to measure viral DNA/RNA levels across a wide dynamic range. The strong correlation observed with the Abbott Alinity m assay suggests that the Hologic Aptima Quant assay can be confidently used to detect and quantify viral DNA/RNA for the diagnosis of viral disease and monitoring of viral load in patients undergoing treatment.

This work was supported by the Dong-A University research fund.