Saliva Proteomics Points to New Biomarker Candidates for Hepatitis B and C

Chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) remains one of the most consequential drivers of liver disease worldwide, underpinning a large share of hepatocellular carcinoma cases, liver cirrhosis, and viral hepatitis-related mortality. Despite decades of progress in antiviral therapy, clinicians still face a persistent diagnostic dilemma: how to detect and monitor liver disease progression without resorting to invasive procedures. A study published in BMC Infectious Diseases by researchers at the Federal University of Rio de Janeiro and collaborators now adds a new dimension to this effort, reporting candidate salivary protein biomarkers that distinguish individuals infected with HBV or HCV from healthy controls.

The research, led by Isabele Batista Campanhon and Lorena Da Rós Gonçalves, who contributed equally to the work, and supervised by corresponding author Márcia Regina Soares, took advantage of an increasingly popular diagnostic fluid: saliva. Over the last two decades, saliva has emerged as a promising medium for disease detection because its collection is noninvasive, painless, and does not require trained phlebotomists or specialized biohazard handling to the same degree as blood. Advances in nanotechnology-based analytical methods have further enabled the detection of low-abundance biomarkers in oral fluid, making it feasible to search for disease signatures in a matrix that was once considered too dilute and too variable for serious clinical proteomics.

To probe the salivary proteome of infected individuals, the team applied a label-free quantification approach coupled with mass spectrometry to whole saliva samples obtained from patients infected with HBV or HCV and from healthy uninfected controls. The analytical platform combined nanoflow liquid chromatography with tandem mass spectrometry (nLC-MS/MS) using a Linear Ion Trap coupled to an Orbitrap mass analyzer (LTQ Orbitrap), an instrument configuration well suited to resolving complex protein mixtures and identifying peptides with high mass accuracy. Label-free strategies avoid the chemical labeling steps used in some quantitative proteomics workflows, reducing sample handling and allowing a larger number of proteins to be compared across groups, though they demand careful statistical treatment of the resulting intensity data.

That statistical treatment is a notable feature of the study’s design and reporting. The authors identified individual candidate protein biomarkers and analyzed them using nominal p values without correction for multiple testing across proteins. This distinction matters: in proteomic experiments that screen thousands of features simultaneously, unadjusted p values can flag proteins as significant by chance alone, and the researchers were explicit that the false discovery rate values reported in their supplementary material relate to protein identification rather than to differential abundance testing. Group comparisons were carried out with nonparametric tests, including Mann–Whitney U tests for pairwise comparisons and Kruskal–Wallis tests across the three study groups, approaches that do not assume normally distributed data and are commonly used for proteomic intensity measurements that often skew across orders of magnitude.

Within this framework, the analysis revealed a distinctive pattern of protein abundance changes in the saliva of infected patients. One protein stood out for its increased expression: IgG Fc-binding protein, which showed higher levels in infected individuals than in healthy controls. This molecule, associated with mucosal surfaces and with the binding of the Fc portion of immunoglobulin G, is an intriguing candidate in the context of chronic viral infection, where systemic and mucosal antibody responses are continuously engaged. Its elevation in the saliva of HBV- and HCV-infected patients suggests that chronic hepatitis may leave a measurable imprint on the oral immune environment, although the authors present these findings as candidate biomarkers requiring further validation rather than as established diagnostic markers.

In contrast to the elevated IgG Fc-binding protein, several secreted proteins were significantly less abundant in the saliva of infected individuals than in healthy controls. The downregulated group included haptoglobin, prolactin-inducible protein, submandibular gland androgen-regulated protein 3, and complement C3. Each of these proteins has plausible connections to the biology of infection and inflammation. Haptoglobin is an acute-phase protein best known for binding free hemoglobin, and complement C3 is a central component of the complement cascade that bridges innate immunity to pathogen clearance. Prolactin-inducible protein, secreted abundantly by the lacrimal and salivary glands, has been implicated in immune regulation at mucosal sites, and submandibular gland androgen-regulated protein 3 reflects the secretory activity of the major salivary glands themselves. A coordinated decrease in such proteins could reflect alterations in glandular function, systemic inflammatory signaling, or the redistribution of immune proteins during chronic viral disease.

The clinical motivation behind the search for salivary markers is grounded in the limitations of current practice. Liver biopsy, although often regarded as the gold standard diagnostic method for assessing hepatic disease, presents important limitations related to safety, cost, and intraobserver and interobserver variability. Sampling error is inherent to a procedure that extracts only a tiny fragment of a large organ, and the interpretation of histological features can differ between pathologists examining the same specimen. Noninvasive alternatives, from serum fibrosis scores to transient elastography, have reduced reliance on biopsy, but reliable biomarkers for early diagnosis and longitudinal disease monitoring remain an unmet need given the severity of HBV- and HCV-associated diseases. Saliva, if validated, could complement these tools with a sample that patients can provide easily and repeatedly.

The study was approved by the Ethics Committee of the Federal University of Rio de Janeiro (protocol 80709 HUCFF/FM/UFRJ), and written informed consent was obtained from all participating patients. The mass spectrometry analyses were supported by the Mass Spectrometry Laboratory at the Brazilian Biosciences National Laboratory (LNBio), CNPEM-ABTLuS, in Campinas, Brazil, and Dr. Yuri Pereira Souza provided bioinformatics support. The work was funded by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant APQ1 E-26/111.770/2012), and Gonçalves received a postdoctoral scholarship from CAPES through its PNPD Institutional Program. The authors declared no competing interests, and the article is published open access under a Creative Commons license.

An important caveat accompanies the findings, and it is one the authors themselves flag in their supplementary documentation: the group-comparison p values were not adjusted for multiple testing across proteins. This means the reported candidates should be viewed as hypotheses generated by an exploratory inter-individual variation study rather than as confirmed diagnostic markers. The inter-individual component of the study’s title is also instructive, acknowledging that protein abundance in saliva varies considerably from person to person, influenced by genetics, diet, oral health, circadian rhythms, and glandular physiology. Any biomarker that survives the transition from discovery to clinical use must demonstrate that the differences between infected and uninfected individuals exceed this background variation, ideally in independent and larger cohorts.

Even with those caveats, the study expands the current understanding of salivary protein profiles associated with HBV and HCV infections and supports the broader proposition that saliva can serve as a noninvasive source of biomarkers for viral hepatitis. As nanotechnology-based detection methods continue to improve sensitivity for low-abundance analytes, candidate proteins such as IgG Fc-binding protein, haptoglobin, prolactin-inducible protein, submandibular gland androgen-regulated protein 3, and complement C3 provide concrete molecular targets for follow-up studies using targeted mass spectrometry or immunoassays. For a disease burden measured in hundreds of millions of chronic infections globally, a simple spit test that could flag liver disease earlier, or track its course without a needle, remains a compelling goal, and this proteomic survey marks a measured but meaningful step along that path.

Subject of Research: Salivary protein biomarker discovery for hepatitis B and C virus infection using label-free mass spectrometry

Article Title: An inter-individual variation study reveals new candidate biomarkers for hepatitis B and C

Article References: Campanhon, I. B., Gonçalves, L. D. R., Sandim, V., Nunes, R. D. O., Melo, A. C. D. A., Moreira, M. F., & Soares, M. R. (2026). An inter-individual variation study reveals new candidate biomarkers for hepatitis B and C. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14449-2

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14449-2

Keywords: hepatitis B, hepatitis C, HBV, HCV, saliva, biomarkers, proteomics, mass spectrometry, label-free quantification, liver disease, viral hepatitis, diagnostics