Rapid, random‐access, and quantification of hepatitis B virus using the Cepheid Xpert HBV viral load assay

Abstract Background Monitoring viral load (VL) is an essential part of the management of patients chronically infected with hepatitis B virus (HBV). The commercial HBV VL assays currently available are generally performed on high‐throughput platforms for batch wise testing of plasma samples, with relatively long turn‐around‐times. Rapid VL testing could provide immediate input to clinical decision making. Methods One hundred two stored plasma samples from 102 patients who were previously tested for HBV VL by the Cobas Ampliprep/Taqman or Cobas 4800 (Roche, Pleasanton, CA), were analyzed by the recently introduced Cepheid Xpert HBV Viral Load Assay. Thirty‐one of the 102 samples were negative for HBV DNA and 71 out of 102 samples had a detectable VL. HBV DNA loads ranged from <20 to 5E8 IU/mL. HBV genotypes (A, B, C, D, E, and G) were known for 52 of the VL positive samples. Correlation of VL results between both assays was determined by the Pearson correlation coefficient (r 2). The level of concordance was assessed using the Bland‐Altman analysis. Results HBV VLs correlated well between both assays, across all genotypes (Pearson correlation coefficient r 2 = 0.987). Six samples exceeded a 0.5  log difference between assays. Bland‐Altman analysis demonstrated a mean of the difference of −0.107 log and a standard deviation of 0.271 log. Conclusion High correlation was observed between the Roche Cobas HBV Viral Load tests and the Xpert HBV Viral Load Assay, thus enabling rapid, random access, and accurate HBV VL assessment.


| Laboratory analysis
The Xpert HBV Viral Load Assay on the GeneXpert random access system is a cartridge based, quantitative molecular test with a limit of quantification of 10 IU/mL and a detection limit of 3.2 IU/mL in plasma, and with a run time of approximately 1 hour and 30 minutes. For the CAP-CTM or C4800, the limit of quantification was 20 IU/mL and the limit of detection was 4.4 IU/mL, with a turnaround time of approximately 6 hours, from the moment the test was started. The input volume for the Xpert HBV Viral Load Assay was 600 µL, the input for the CAP-CTM/C4800 was 400 µL.

| Data analysis
The results obtained by both tests were translated from HBV DNA IU/mL to log IU/mL for further statistical analysis. In positive samples where only one of the measurements fell outside of the measurement range, the measurement was set at the lowest or highest measurable point (eg, (CAP/CTM/C4800 >1.7E8 IU/mL becomes 1.7E8 IU/mL and <20 IU/mL becomes 20 IU/mL for data analysis purposes. The differences in VL between the assays were expressed as log difference. The acceptance criteria for inter-assay differences were set at 0.5 log. The correlation of the results of both assays was determined by calculating the Pearson correlation coefficient (r 2 ).
The level of concordance was assessed using Bland-Altman analysis.
The mean of the differences between the two tests was calculated and plotted against the mean of the measurements. The mean and the standard deviation (SD) of all log translated VL results were calculated using a one-sample t test. The 95% limits of agreement were determined as the mean ± 1.96 SD.
All calculations were done using SPSS statistics version 25 (IBM, New York).

| RESULTS
A total 102 samples from the original 106 samples were tested in this study. Three cartridge errors and three sample errors occurred, resulting in four samples that could not be repeated due to insufficient material and were therefore not included in the analysis. Thirty-one samples previously tested negative on the CAP/CTM or C4800 for HBV DNA. Two of these samples had a VL of <10 IU/mL on the GeneXpert, the remaining samples   (Table 1).
All results for QCMD samples that were run on the GeneXpert were within 0.5 log difference of the consensus result reported by QCMD, the largest difference being 0.210 log.

| DISCUSSION
Correlation and agreement between the routine laboratory assays (CAP-CTM and C4800) and the Xpert HBV Viral Load assay was evaluated for 102 samples previously tested on CAP-CTM or C4800.
A high agreement was observed between HBV VL results generated in routine laboratory assays (CAP-CTM and C4800) and the Xpert HBV Viral Load assay across all tested genotypes. The quantification of the HBV DNA concentration in the sample is done by using high and low internal quantitative standards in tandem with specific acceptance criteria. Additionally, these internal quantitative standards are used as controls to detect specimenassociated inhibition of the PCR reaction.
The pretest preparations take less than 10 minutes. The turnaround time of the Xpert HBV Viral Load assay was approximately an hour. After the test has reached completion, the results need to be checked and confirmed before they can be conveyed to the clinician. Therefore, the total time from the cartridge preparation to the release the VL results, is approximately 1 hour and 30 minutes.
The random-access nature and fast assay turnaround time of the GeneXpert changes the dynamics and routing of HBV VL testing in the routine microbiological laboratory and leads to a more efficient laboratory workflow and a significant improvement in speed in which the result is available for patient care.
In conclusion high agreement and correlation were observed between both HBV viral load assays with regard to the HBV genotypes tested. The Xpert HBV Viral Load assay is a user-friendly random-access test for rapid, accurate virological assessment of HBV infected patients.

CONFLICT OF INTERESTS
None of the contributing authors have any conflicts of interest, except that the cartridges used in the viral load testing of Hepatitis B virus on the GeneXpert instrument System (Cepheid, Sunnyvale, CA) were provided by Cepheid.

AUTHOR CONTRIBUTIONS
AMA, SS, and CP performed testing. AMA analyzed the data and wrote the manuscript with input from all authors.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.