# Rapid test for hepatitis B core-related antigen to identify people living with hepatitis B having high viral load in Cameroon

Richard Njouom, Alassane Ndiaye, Abdou Fatawou Modiyinji, Frederic Lissock, Jeanne Perpétue Vincent, Masaya Baba, Naoki Yamamoto, Atsushi Kaneko, Katsumi Aoyagi, Naofumi Hashimoto, et al.

► To cite this version:

Richard Njouom, Alassane Ndiaye, Abdou Fatawou Modiyinji, Frederic Lissock, Jeanne Perpétue Vincent, et al. Rapid test for hepatitis B core-related antigen to identify people living with hepatitis B having high viral load in Cameroon. Virology, 2025, 602, pp.110316. 10.1016/j.virol.2024.110316. pasteur-05281364

HAL Id: pasteur-05281364

https://pasteur.hal.science/pasteur-05281364v1

Submitted on 24 Sep 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Rapid test for hepatitis B core-related antigen to identify people living with hepatitis B having high viral load in Cameroon

Richard Njouom<sup>a, **</sup>, Alassane Ndiaye<sup>b</sup>, Abdou Fatawou Modiyinji<sup>a</sup>, Frederic Lissock<sup>a</sup>, Jeanne Perpétue Vincent<sup>b</sup>, Masaya Baba<sup>c</sup>, Naoki Yamamoto<sup>d</sup>, Atsushi Kaneko<sup>d</sup>, Katsumi Aoyagi<sup>d</sup>, Naofumi Hashimoto<sup>e</sup>, Mari Nagai<sup>e</sup>, Masato Ichikawa<sup>f</sup>, Tetsuo Miura<sup>f</sup>, Wataru Sugiura<sup>f,g</sup>, Yasuhito Tanaka<sup>g,h</sup>, Yusuke Shimakawa<sup>b,c,g,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Handling Editor: Dr. Jasmine Tomar

**Keywords:**

Hepatitis B core-related antigen

Point-of-care tests

Validation studies

Sensitivity and specificity

Africa

Elimination

## ABSTRACT

This study presents a retrospective assessment of the diagnostic performance of the newly developed hepatitis B core-related antigen rapid diagnostic test (HBcrAg-RDT) in detecting plasma samples with elevated hepatitis B virus (HBV) DNA levels (≥200,000 IU/ml) in Yaoundé, Cameroon. Samples were collected consecutively from treatment-naïve adults living with HBV between January 1, 2021, and June 30, 2023. Analyzing 146 samples from participants with a median age of 36 years, the HBcrAg-RDT exhibited a sensitivity of 97.5% (95% CI: 86.8–99.9) and a specificity of 77.4% (68.2–84.9) when compared to real-time PCR as the reference standard. These findings suggest that HBcrAg-RDT holds promise as a valuable point-of-care tool for diagnosing high HBV DNA levels, particularly in resource-limited settings. Further research will refine its practicality and effectiveness.

## 1. Introduction

The global elimination of the hepatitis B virus (HBV) necessitates the expansion of testing and treatment services in low- and middle-income countries (LMICs). Following the screening for hepatitis B surface antigen (HBsAg), the quantification of serum HBV DNA has been recommended to identify a subset of people living with HBV (PLWHB) who should commence antiviral therapy. To prevent mother-to-child transmission (MTCT) of HBV, antiviral prophylaxis is recommended for HBsAg-positive pregnant women with high viremia (≥200,000 IU/ml or ≥5.3 log10 IU/mL) (Terrault et al., 2018; European Association and for the Study of the Liver, 2017; WHO, 2020). Despite its central role in the clinical management of chronic HBV infection (CHB), over 95% of HBV-infected individuals reside in LMICs (Razavi-Shearer et al., 2023)

where HBV DNA quantification is not easily accessible.

Hepatitis B core-related antigen (HBcrAg) assay quantifies total levels of particle-free hepatitis B e antigen (HBeAg), p22 core-related protein (PreC), hepatitis B core antigen (HBcAg) in DNA-containing particles, and phosphorylated HBcAg in empty or RNA-containing particles, using a chemiluminescence immunoassay (CLIA) (Kimura et al., 2005; Hong et al., 2021; Inoue et al., 2021, 2023). This marker has emerged as a promising alternative for HBV DNA quantification in treatment-naïve PLWHB due to its close correlation with intrahepatic covalently closed circular DNA (cccDNA) levels and serum HBV DNA levels (Mak et al., 2018; Maasoumy et al., 2015; Testoni et al., 2019; Yoshida et al., 2021). Indeed, maternal HBcrAg levels during pregnancy accurately predicted MTCT events despite infant immunization (Shimakawa et al., 2022). We recently developed a rapid diagnostic test

<footnote><sup>a</sup> Department of Virology, Centre Pasteur of Cameroon, Yaoundé, Cameroon</footnote>

<footnote><sup>b</sup> Institut Pasteur, Université Paris Cité, Unité d'Épidémiologie des Maladies Émergentes, Paris, France</footnote>

<footnote><sup>c</sup> International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan</footnote>

<footnote><sup>d</sup> Research and Development Division, Fujirebio Inc., Tokyo, Japan</footnote>

<footnote><sup>e</sup> Bureau of International Health Cooperation, National Center for Global Health and Medicine, Tokyo, Japan</footnote>

<footnote><sup>f</sup> Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan</footnote>

<footnote><sup>g</sup> Pasteur International Unit at Kumamoto University / National Center for Global Health and Medicine, Japan</footnote>

<footnote><sup>h</sup> Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan</footnote>

<footnote>* Corresponding author. Institut Pasteur, Université Paris Cité, Unité d'Épidémiologie des Maladies Émergentes, Paris, France.</footnote>

<footnote>** Corresponding author. Department of Virology, Centre Pasteur of Cameroon, Yaoundé, Cameroon.</footnote>

<footnote>E-mail addresses: njouom@pasteur-yaounde.org (R. Njouom), yusuke.shimakawa@gmail.com (Y. Shimakawa).</footnote>
based on immunochromatography that enables the detection of HBcrAg (HBcrAg-RDT) at the point of care (Shimakawa et al., 2023). HBcrAg-RDT is a lateral flow assay using anti-HBcrAg monoclonal antibodies. A test kit contains a single-use disposable plastic cassette, pretreatment solution, neutralizing solution, squeeze tube, and applicator tip. For 50 µL of serum, plasma, or whole blood, 150 µL of pretreatment solution and 50 µL of neutralizing solution are required. No special equipment, nor electricity, is required.

We previously reported on the performance of the HBcrAg-RDT in 284 treatment-naïve PLWHB from the PROLIFICA cohort in The Gambia, west Africa (Shimakawa et al., 2023). Using stored sera (–80 °C) sent to Toshiba General Hospital, Japan, the sensitivity and specificity of HBcrAg-RDT to diagnose clinically important HBV DNA levels (≥5.3 log10 IU/ml) were 91.4% and 86.3%, respectively. While the study provided promising results, a limitation was that the test was conducted in a laboratory in Japan. There is a need to validate these findings in laboratories located in LMICs. Here, we report the performance of HBcrAg-RDT evaluated at the Centre Pasteur of Cameroon in Yaoundé, Cameroon.

## 2. Materials and methods

We retrospectively evaluated the performance of HBcrAg-RDT to diagnose high HBV DNA levels (≥5.3 log10 IU/mL) using plasma from treatment-naïve adult PLWHB, collected between January 1, 2021, and June 30, 2023, and stored at –80 °C at the Centre Pasteur of Cameroon in Yaoundé. We used real-time PCR (Abbott RealTime HBV Assay, Illinois, USA, limit of detection: 10 IU/mL) as a reference. HBcrAg-RDT was performed by a laboratory technician blinded to the results of HBV DNA levels. The study protocol was approved by the Institutional Review Board of Kumamoto University (ref: Senshin 1094) and the Ethics Committee in Cameroon (ref: N° 2015/04/524 CNERSH/SP).

## 3. Results

A total of 146 individuals with chronic HBV infection participated in the study. Their median age was 36 years (interquartile range: 29–44), and 86 (58.9%) were male. The distribution of HBV DNA levels (log10 IU/mL) was as follows: <3.3 (n = 42, 28.8%), 3.3–4.2 (n = 27, 18.5%), 4.3–5.2 (n = 37, 25.3%), 5.3–6.2 (n = 9, 6.2%), 6.3–7.2 (n = 8, 5.5%), 7.3–8.2 (n = 16, 11.0%), and ≥8.3 (n = 7, 4.8%). Using plasma stored at –80 °C, 63 (43.2%) were positive for HBcrAg-RDT, 79 (54.1%) were negative for HBcrAg-RDT, and 4 (2.7%) had invalid result. Samples initially yielding invalid results underwent retesting using a new reaction cassette, all of which returned negative results upon reevaluation. The sensitivity and specificity of HBcrAg-RDT to diagnose high HBV DNA levels (≥5.3 log10 IU/ml) were 97.5% (95% CI: 86.8–99.9) and 77.4% (68.2–84.9), respectively. The figure presents the proportion of samples tested positive for HBcrAg-RDT across different HBV DNA levels (see Fig. 1).

## 4. Discussion

We retrospectively evaluated a novel point-of-care test for detecting high viremia in plasma at a laboratory located in LMICs. Its low production cost, simple specimen preparation, lack of requirement for electricity or equipment, operating temperature (up to 39 °C), and rapid turnaround time (45 min) all favour its use at the point of care in LMICs. The shelf life is 12 months, and the test can be stored at 2–30 °C until expiration or up to 50 °C for up to two weeks. In areas where access to HBV DNA quantification is limited, the World Health Organization (WHO) recommends HBeAg testing as an alternative to HBV DNA to determine eligibility for peripartum antiviral prophylaxis (WHO, 2020). This recommendation was based on a systematic review and meta-analysis of HBeAg performance showing a pooled sensitivity of 88.2% and specificity of 92.6% for indicating viral loads ≥5.3 log10

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Proportion of HBcrAg-RDT positive samples at different HBV DNA levels.</figcaption></figure>

IU/mL (Boucheron et al., 2021). However, these estimates were mostly based on studies using laboratory-based immunoassays such as enzyme immunoassay (EIA) or chemiluminescent immunoassay (CLEIA). In studies using rapid diagnostic test (RDT) for HBeAg, the pooled sensitivity was only 70.1% (Boucheron et al., 2021). The low diagnostic sensitivity of HBeAg RDT is most likely attributable to a limited analytical sensitivity of these RDTs compared to the laboratory-based HBeAg immunoassays (Seck et al., 2018; Ségeral et al., 2018; Stockdale et al., 2021). Compared to these, the performance of HBcrAg-RDT observed in this study (sensitivity 97.5%, specificity 77.4%) was highly promising.

As with any novel diagnostic tool, limitations exist. HBcrAg-RDT appears useful for initiating antiviral therapy for pregnant women but may not be suitable for monitoring treatment response. Additionally, our study has limitations. First, validation was performed using stored plasma from a single cohort, necessitating further evaluation in diverse cohorts, especially using different sample types, such as capillary whole blood. Second, we did not evaluate viral genotypes. However, it is well established that in Cameroon both HBV genotypes A and E are prevalent (Atsama Amougou et al., 2019). Third, we were unable to compare the performance of HBcrAg-RDT with that of HBeAg in this study.

## 5. Conclusions

In conclusion, HBcrAg-RDT exhibits promise as a valuable tool in identifying individuals having a high viral load in LMICs. Further studies will refine its applicability and assess its potential contribution to global HBV elimination efforts, as well as strengthening diagnostic capacity globally (World Health Organization and (WHO), 2023).

### CRediT authorship contribution statement

**Richard Njouom:** Writing – original draft, Conceptualization. **Alassane Ndiaye:** Writing – original draft, Formal analysis. **Abdou Fatawou Modiinyinji:** Data curation. **Frederic Lissock:** Resources. **Jeanne Perpétue Vincent:** Writing – review & editing, Funding acquisition. **Masaya Baba:** Resources. **Naoki Yamamoto:** Resources. **Atsushi Kaneko:** Resources. **Katsumi Aoyagi:** Resources. **Naofumi Hashimoto:** Supervision. **Mari Nagai:** Supervision. **Masato Ichikawa:** Supervision. **Tetsuo Miura:** Supervision. **Wataru Sugiura:** Supervision. **Yasuhito Tanaka:** Writing – review & editing, Supervision, Conceptualization. **Yusuke Shimakawa:** Writing – review & editing, Writing – original draft, Project administration, Funding acquisition, Conceptualization.
## Data sharing

Individual participant data will not be made publicly available, since data contain protected health information. However, deidentified participant data may be shared upon reasonable request if this was approved by the Ethics committee in Cameroon.

## Financial Support

This work was supported by the JSPS KAKENHI Grant Number JP21K10416, the grant-in-aid from the Research Program on Hepatitis from the Japan Agency for Medical Research and Development (AMED JP23fk0310518), and the Pasteur International Unit at Kumamoto University/National Center for Global Health and Medicine, Japan.

## Declaration of competing interest

NY, AK, and KA are employees of Fujirebio Inc. YT received research grants from Gilead Sciences, Sysmex Corp, GlaxoSmithKline PLC, Fujirebio Inc, and Janssen Parmaceutical K.K. YS received research grants from Gilead Sciences and research materials from Abbott and Fujirebio Inc. The other authors declare no conflict of interest.

## Acknowledgements

We acknowledge Paul Franck Adeyissimi Adjou Moumouni, Akira Hasegawa, Kazuyoshi Miyamoto, Tomonao Ouki, Takehisa Watanabe, and Shintaro Yagi their support to conduct the project.

## References

* Atsama Amougou, M., Marchio, A., Bivigou-Mboumba, B., et al., 2019. Enrichment in selected genotypes, basal core and precore mutations of hepatitis B virus in patients with hepatocellular carcinoma in Cameroon. J. Viral Hepat. 26, 1086-1093.

* Boucheron, P., Lu, Y., Yoshida, K., et al., 2021. Accuracy of HBeAg to identify pregnant women at risk of transmitting hepatitis B virus to their neonates: a systematic review and meta-analysis. Lancet Infect. Dis. 21, 85-96.

* European Association for the Study of the Liver, 2017. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J. Hepatol. 67, 370-398.

* Hong, X., Luckenbaugh, L., Mendenhall, M., et al., 2021. Characterization of hepatitis B precore/core-related antigens. J. Virol. 95. https://doi.org/10.1128/jvi.01695-20.

* Inoue, T., Kusumoto, S., Iio, E., Ogawa, S., Suzuki, T., Yagi, S., Kaneko, A., Matsuura, K., Aoyagi, K., Tanaka, Y., 2021. Clinical efficacy of a novel, high-sensitivity HBcrAg assay in the management of chronic hepatitis B and HBV reactivation. J. Hepatol. 75 (2), 302-310.

* Inoue, T., Watanabe, T., Tanaka, Y., 2023. Hepatitis B core-related antigen: A novel and promising surrogate biomarker to guide anti-hepatitis B virus therapy. Clin. Mol. Hepatol. 29 (4), 851-868. https://doi.org/10.3350/cmh.2022.0434.

* Kimura, T., Ohno, N., Terada, N., et al., 2005. Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain. J. Biol. Chem. 280, 21713-21719.

* Maasoumy, B., Wiegand, S.B., Jaroszewicz, J., et al., 2015. Hepatitis B core-related antigen (HBcrAg) levels in the natural history of hepatitis B virus infection in a large European cohort predominantly infected with genotypes A and D. Clin. Microbiol. Infection 21, 606.e1-606.e10.

* Mak, L.Y., Wong, D.K.H., Cheung, K.S., Seto, W.K., Lai, C.L., Yuen, M.F., 2018. Review article: hepatitis B core-related antigen (HBcrAg): an emerging marker for chronic hepatitis B virus infection. Aliment. Pharmacol. Ther. 47, 43-54.

* Razavi-Shearer, D., Gamkrelidze, I., Pan, C., et al., 2023. Global prevalence, cascade of care, and prophylaxis coverage of hepatitis B in 2022: a modelling study. The Lancet Gastroenterology and Hepatology 8, 879-907.

* Seck, A., Ndiaye, F., Maylin, S., et al., 2018. Poor Sensitivity of Commercial Rapid Diagnostic Tests for Hepatitis B e Antigen in Senegal, West Africa. Am. J. Trop. Med. Hyg. 99, 428-434.

* Ségéral, O., N'Diaye, D.S., Prak, S., et al., 2018. Usefulness of a serial algorithm of HBsAg and HBeAg rapid diagnosis tests to detect pregnant women at risk of HBV mother-to-child transmission in Cambodia, the ANRS 12328 pilot study. J. Clin. Virol. 109, 29-34.

* Shimakawa, Y., Veillon, P., Birguel, J., et al., 2022. Residual risk of mother-to-child transmission of hepatitis B virus infection despite timely birth-dose vaccination in Cameroon (ANRS 12303): a single-centre, longitudinal observational study. Lancet Global Health 10, 1-9.

* Shimakawa, Y., N dow, G., Kaneko, A., et al., 2023. Rapid point-of-care test for hepatitis B core-related antigen to diagnose high viral load in resource-limited settings. Clin. Gastroenterol. Hepatol. 21, 1943-1946.e2.

* Stockdale, A.J., Silungwe, N.M., Shawa, I.T., Kreuels, B., Gordon, M.A., Geretti, A.M., 2021. Diagnostic performance evaluation of hepatitis B e antigen rapid diagnostic tests in Malawi. BMC Infect. Dis. 21, 1-10.

* Terrault, N.A., Lok, A.S.F., McMahon, B.J., et al., 2018. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 67, 1560-1599. Baltimore, Md.

* Testoni, B., Lebossé, F., Scholtes, C., et al., 2019. Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients. J. Hepatol. 70, 615-625.

* WHO, 2020. Prevention of Mother-To-Child Transmission of Hepatitis B Virus: Guidelines on Antiviral Prophylaxis in Pregnancy. Geneva, Switzerland.

* World Health Organization (WHO), 2023. WHA 76.5: Strengthening Diagnostics Capacity.

* Yoshida, K., Desbiolles, A., Feldman, S.F., et al., 2021. Hepatitis B core-related antigen to indicate high viral load: systematic review and meta-analysis of 10,397 individual participants. Clin. Gastroenterol. Hepatol. 19, 46-60.e8.