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Selective detection of HBV pre-genomic RNA in chronic hepatitis B patients using a novel RT-PCR assay

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Abstract

In chronic hepatitis B (CHB) patients, quantification of HBV pgRNA in plasma has the potential to provide information on disease prognosis and liver injury or histopathology. However, current methods for detecting HBV pgRNA present technical difficulties due to the co-existence of HBV DNA in plasma samples. We have successfully established a novel one-step RT-PCR assay that allows selective quantification of HBV pgRNA. Two cohorts of participants were recruited for assay validation, including treatment-naïve patients with CHB and HBeAg-positive CHB patients who were treated with Tenofovir and monitored for 6 months to assess the predictive value of baseline HBV RNA for HBeAg seroclearance. Statistical analysis was performed using MedCalc version 20.019 software. The novel selective one-step RT-PCR assay for detecting HBV pgRNA was validated with a limit of detection of 100 copies/mL. The assay was able to selectively measure HBV pgRNA even in the presence of excess HBV rcDNA. In treatment-naïve CHB patients, HBV pgRNA levels were significantly lower than HBV DNA concentration. Serum HBV DNA levels and HBeAg status were positively associated with HBV pgRNA. Baseline serum HBV pgRNA levels were found to be strong predictors of HBeAg seroclearance after 6 months of Tenofovir treatment. The study presents a novel RT-PCR assay that allows accurate measurement of plasma HBV pgRNA in chronic hepatitis B patients, even in the presence of excess HBV DNA. The assay is highly selective and represents a significant advancement with potential for further breakthroughs in understanding the clinical significance of HBV pgRNA.

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Acknowledgements

We thank Tai Pham, Tien Tran, Hung Hoang for their valuable discussion and input, Quyen Pham, Ly Vu, Ly Hoang, Toan Vu, Quyen Nguyen, Hang Pham for their exceptional technical assistance. This work was supported by the Hanoi Department of Science & Technology (grant numbers 01C-08/08-2017-3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funding

This work was supported by the Hanoi Department of Science & Technology (grant numbers 01C-08/08–2017-3).

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Author notes

  1. Ung Dinh Nguyen and Quyen Le Do have contributed equally to this work.

Authors and Affiliations

  1. Department of Genomics and Cytogenetics, Institute of Biomedicine and Pharmacy (IBP), Vietnam Military Medical University, Hanoi, Vietnam

    Ung Dinh Nguyen, Quynh Anh Nguyen Vu, Nguyet Thi Trieu, Trang Thuy Dao & Tho Huu Ho

  2. Department of Infectious Disease, 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam

    Quyen Le Do, Nam Van Le, Tuyen Tien Hoang, Chinh Trong Nguyen & Dien Van Nguyen

  3. Department of Microbiology, 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam

    Son Thai Nguyen & Tho Huu Ho

  4. Medical Testing Center, Medlatec Group, Hanoi, Vietnam

    Son Thai Nguyen

  5. Outpatient Department, 103 Military Hospital, Vietnam Military Medical University, Hanoi, Vietnam

    Thang Hong Nguyen

  6. Systems Immunity Research Institute, School of Medicine, Cardiff University, Cardiff, UK

    Dien Van Nguyen

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  1. Ung Dinh Nguyen
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by UDN, QLeD, QANV, NTT, TTD, NVanL, STN, TTH, CTN, THN, THH. The first draft of the manuscript was written by UDN, QLeD, TTD, THH and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Tho Huu Ho.

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Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee of Vietnam Military Medical University (reference number: 780/QĐ-HVQY, dated 28/03/2018) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

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Nguyen, U.D., Le Do, Q., Vu, Q.A.N. et al. Selective detection of HBV pre-genomic RNA in chronic hepatitis B patients using a novel RT-PCR assay. Clin Exp Med 23, 5281–5289 (2023). https://doi.org/10.1007/s10238-023-01162-6

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  • DOI: https://doi.org/10.1007/s10238-023-01162-6

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