Uncovering silent carriers: Hematological insights and viral burden in incidentally detected hepatitis B virus infection

Abstract

BACKGROUND

Hepatitis B Virus (HBV) remains a global public health challenge, affecting over 296 million people, many of whom are asymptomatic. Incidentally diagnosed carriers provide a critical window for early intervention and prevention. Understanding their hematological profile and viral burden can help inform risk assessment and clinical management.

AIM

To evaluate hematological parameters and HBV viral load in incidentally detected asymptomatic hepatitis B surface antigen positive patients during routine health screenings.

METHODS

A cross-sectional observational study was conducted from June 2024 to March 2025 at Dr. D. Y. Patil Medical College, Hospital and Research Centre a tertiary care hospital in Pune, Maharashtra, India, involving 100 hepatitis B surface antigen-positive patients and 20 healthy controls. Hematological and liver function parameters were assessed, and quantitative HBV DNA analysis was performed using real-time polymerase chain reaction. Statistical analysis was conducted using GraphPad Prism v8.0.

RESULTS

Marked variations were detected in hemoglobin levels (P < 0.0001), percentage of neutrophils (P = 0.0006), percentage of lymphocytes (P = 0.0031), serum glutamic-oxaloacetic transaminase activity (P = 0.0013), and alanine aminotransferase levels (P = 0.0001) when comparing HBV-infected individuals with the control group. Conversely, differences in total leukocyte count, percentage of monocytes, percentage of eosinophils, platelet count, total bilirubin, and serum glutamic-pyruvic transaminase values were not statistically significant. Viral load > 2000 IU/mL was found in 17 patients, and < 2000 IU/mL in 24 patients. Viral load positively correlated with conjugated bilirubin, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, and alkaline phosphatase, and negatively with albumin and the albumin-globulin ratio.

CONCLUSION

Incidentally detected HBV infections present an opportunity for early disease detection. Hematological and viral markers can guide clinical decisions. Routine screening and contact tracing are essential strategies to control HBV transmission and progression.

Key Words: Hepatitis B; Hepatitis B virus deoxyribonucleic acid quantification; Hematological biomarkers; Liver function tests; Viral load; Subclinical hepatic injury

Core Tip: A substantial proportion of hepatitis B virus infections remain undetected due to the asymptomatic nature of silent carriers. This study reveals that incidentally diagnosed hepatitis B surface antigen-positive individuals exhibit significant alterations in hematological and liver function parameters despite lacking clinical symptoms. Findings demonstrate a correlation between viral load and markers of hepatic injury, highlighting the need for vigilant laboratory monitoring. Early identification of subclinical liver involvement offers a critical window for intervention to prevent long-term complications such as cirrhosis and hepatocellular carcinoma. This work underscores the importance of integrating routine screening, hematological profiling, and virological assessment in public health strategies targeting hepatitis B virus control.

INTRODUCTION

Infection with the hepatitis B virus (HBV) is a major worldwide health risk. The World Health Organization recently estimated that 296 million individuals globally suffer from a persistent HBV infection[1,2]. Despite its high prevalence, a substantial proportion of infected individuals remain asymptomatic for prolonged periods, unknowingly carrying and potentially transmitting the virus to others. These individuals often referred to as silent or asymptomatic carriers are typically identified during routine health check-ups, preoperative evaluations, or investigations for unrelated medical conditions[3].

The clinical implications of asymptomatic HBV carriage are profound. Although these individuals may not exhibit overt signs of liver dysfunction, the virus may still be actively replicating within hepatocytes, causing subclinical inflammation and progressive liver damage[4]. If left undetected and untreated, this can eventually lead to serious complications, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Moreover, asymptomatic carriers contribute silently to the transmission chain, representing a hidden reservoir that undermines efforts to control and eliminate HBV at the community and global levels[5].

Timely detection of these individuals through routine screening programs offers a crucial opportunity to intervene early in the disease course[6]. By identifying carriers before clinical symptoms develop, healthcare providers can initiate appropriate monitoring strategies, consider antiviral therapy when indicated, and implement measures to reduce the risk of transmission such as educating patients, tracing and screening contacts, and promoting vaccination[7].

Despite the known importance of early identification, there exists a significant gap in the literature concerning the hematological and virological profiles of these incidentally diagnosed HBV carriers[8]. Specifically, the relationship between viral load and blood-based biomarkers such as hemoglobin levels, white blood cell count, liver enzymes, and serum proteins remains underexplored in asymptomatic populations[9,10].

The current study was designed to address this gap. By systematically analyzing the hematological parameters and HBV DNA levels in patients incidentally found to be hepatitis B surface antigen (HBsAg)-positive, we aim to gain insights into early, subclinical alterations that may signify active viral replication or impending liver dysfunction[11]. The findings of this research may contribute to refining risk stratification models, enhancing clinical surveillance protocols, and improving public health strategies targeting the silent burden of HBV[12].

MATERIALS AND METHODS

Study design and setting

The current study was conducted at Dr. D. Y. Patil Medical College, a tertiary healthcare facility in Pune, Maharashtra, India, along with its affiliated hospital and research center, over a ten-month period, from June 2024 to March 2025. It was designed as a cross-sectional qualitative investigation. Assessing and contrasting the virological, biochemical, and haematological characteristics of people who were found to be asymptomatic carriers of the HBsAg with that of twenty healthy persons who were found to be HBV-free was the main goal. The study site offered a heterogeneous patient population and access to advanced diagnostic infrastructure, ensuring thorough assessments and dependable data acquisition. The required sample size was calculated considering an expected population proportion of 2%, an absolute precision of 3%, and a 95% confidence level. After adjusting for an anticipated non-participation rate of 20%, the final sample size was determined to be 100 subjects.

Study population

Participants were grouped into two distinct categories: (1) HBV group (n = 100): This group consisted of individuals who were incidentally identified as HBsAg-positive during their clinical evaluations. These participants showed no signs or symptoms of hepatitis at the time of diagnosis and were recruited from outpatient/inpatients departments and routine check-ups; and (2) Healthy control group (n = 20): This group comprised age- and gender-matched healthy volunteers who were confirmed negative for HBV markers through serological testing. These individuals had no prior history of liver-related illness, chronic disease, or immunosuppressive conditions and served as a comparative baseline for laboratory parameters.

Inclusion criteria

Participants were eligible to take a part in the investigation provided that they met the conditions that followed: (1) Age: 18 years or above, to ensure the inclusion of adults with comparable physiological characteristics; and (2) Health status: Clinically asymptomatic persons who were identified as HBsAg-positive through routine preoperative screening or general health examinations.

Exclusion criteria

To ensure data integrity and avoid confounding effects, individuals were excluded from the study if they had any of the following conditions: (1) Presence of hepatitis C virus or human immunodeficiency virus co-infection, verified through standard diagnostic procedures; and (2) Immunocompromised states, whether due to congenital immunodeficiencies, chronic illnesses, or ongoing immunosuppressive therapy.

Data collection

Patient case records were reviewed retrospectively to collect demographic and clinical information. The following parameters were recorded.

Demographic information: Including age, sex, and duration of hospital admission (where applicable).

Laboratory parameters: (1) Haematological tests: Haemoglobin levels, total leukocyte count, differential leukocyte count, and platelet count; (2) Biochemical tests: Liver function tests such as serum glutamic-oxaloacetic transaminase (SGOT) or aspartate aminotransferase (AST), serum glutamic-pyruvic transaminase (SGPT) or alanine aminotransferase (ALT), alkaline phosphatase (ALP), total and direct bilirubin, and serum albumin; and (3) Molecular tests: Quantitative estimation of HBV DNA viral load was performed for HBsAg-positive cases using a real-time polymerase chain reaction (PCR) method.

Laboratory investigations

All laboratory analysis were performed in compliance with established protocols approved by National Accreditation Board for Testing and Calibration Laboratories, ensuring accuracy, precision, and quality control in diagnostic testing. The investigations encompassed serological, biochemical, haematological, and molecular assessments as outlined below.

Serological testing for HBsAg: Serum specimens were analyzed for the presence of HBsAg using the Architect HBsAg assay kit on the Abbott ARCHITECT ci8200 platform (Abbott Ireland Diagnostics Division, Lisnamuck, Longford, Ireland). The assay employs a chemiluminescent microparticle immunoassay technique. As per the manufacturer’s guidelines, samples with HBsAg concentrations ≥ 0.05 IU/mL were interpreted as reactive. The method is characterized by high analytical sensitivity and specificity, reducing the likelihood of false-positive results and enabling accurate detection of HBV carriers[13,14].

Biochemical analysis - liver function tests: Evaluation of hepatic function was conducted through the measurement of routine liver function test parameters using Architect C8000 system. Serum samples were used to assess enzyme activity and protein levels reflective of hepatic cellular integrity, biliary function, and synthetic capacity using the commercially available Abbott laboratories kits. The key enzymes analyzed were AST/SGOT, ALT/SGPT, total bilirubin and direct (conjugated) bilirubin levels were determined. The ranges over which results were reported were as per the manufacturer’s instructions. All test results were reported in standard international units, and values were interpreted with reference to established normal ranges.

Both aminotransferases are intracellular enzymes predominantly found in hepatocytes, and their elevation in serum indicates hepatocellular injury, inflammation, or necrosis, which are common in viral hepatitis[15]. ALP was measured as a marker of biliary tract health. Elevated ALP levels often suggest cholestasis or intrahepatic bile duct obstruction. Additionally, total bilirubin and direct (conjugated) bilirubin levels were determined. Bilirubin is a byproduct of haemoglobin breakdown and serves as an indicator of hepatic excretory function. An increase in total or direct bilirubin levels can reflect impaired bilirubin metabolism or bile flow obstruction. Finally, serum albumin was measured to assess the synthetic function of the liver. As albumin is produced exclusively by hepatocytes, decreased levels may signal chronic liver disease or reduced protein synthesis due to hepatic dysfunction[16,17]. These biochemical parameters were essential in evaluating liver health and correlating with virological and haematological findings across the study population.

Haematological analysis: Haematological profiling was performed to assess general systemic health and to identify any alterations in blood cell parameters that may be associated with liver dysfunction or viral infection. DXH900 Hematology analyzer (Beckman Coulter, Chaska, MN, United States) was used to determine complete blood count. One of the primary parameters evaluated was haemoglobin concentration, which provides an estimate of the oxygen-carrying capacity of blood. A reduced haemoglobin level can indicate anemia, which is frequently observed in chronic illnesses and nutritional deficiencies, both of which may coexist in hepatitis B-infected individuals[18,19].

Immune status of the subjects was evaluated by determining the total white blood cell count and the differential leukocyte count. Alterations in the white blood cell count or in specific leukocyte subsets (such as neutrophils, lymphocytes, or eosinophils) can reflect underlying inflammatory, infectious, or immunological processes. Furthermore, platelet counts were analyzed as thrombocytopenia is a common haematological manifestation in chronic liver disease, often due to hypersplenism or impaired thrombopoietin production[20]. The haematological data were interpreted alongside biochemical and molecular parameters to provide a comprehensive clinical assessment of both HBV-infected patients and healthy controls.

Quantitative estimation of HBV DNA viral load by real time PCR: Plasma samples were assessed for viral load quantification. QuantStudio 5 (Applied Biosystems, CA, United States) Real time PCR system was used. The chemistry used for this PCR was Taqman probe chemistry. TRUPCR total nucleic acid extraction and HBV viral load kits (M/s 3B BlackBio Dx Ltd., Bhopal, India) were used. To ensure the quality of the extracted DNA from clinical samples, an endogenous control gene was included in the kit. Real time PCR protocol, program set up, channel selection, preparation of standards and result analysis were done as per the manufacturer’s instructions. The assay runs for 40 cycles however no amplication beyond 38 cycles was considered for any interpretation hence cut-off of 38 cycles threshold was set by the manufacturer.

Statistical analysis

GraphPad Prism (Version 8.0.1) and SPSS software (Version 26.0) were used for the analysis of the data. The data collection’s uniformity was assessed using the Shapiro-Wilk test. Regarding patients demographic data, statistical measures were computed, such as frequencies and percentages. The Mann-Whitney U test was used for analyses with data that is continuous. Continuous variables, including hematological and biochemical parameters as well as viral load values, were analyzed using the Mann-Whitney U-test. Spearman’s rank correlation analysis was used to look at connections between the variables. A statistically significant P value was defined as a value below 0.05.

RESULTS

Demographic profile

Analysis of the demographic data showed that individuals in the HBV-positive group had an average age of 39.6 ± 12.4 years, placing the majority within the middle-aged category. There was a clear predominance of males, with an approximate male-to-female ratio of 1.8:1. The length of hospital stay was comparable between the HBV-positive and control groups, indicating that asymptomatic HBV infection, in the absence of evident liver-related complications, did not significantly prolong hospitalization (Figure 1A). Enrolled patients were classified based on their residing areas into urban and rural (Figure 1B). Higher infection rates are made possible by urban crowding, frequent migrations and gaps in vaccination coverage among transient populations which may create conditions that favor viral spread, while lower HBV transmission in rural areas may be a result of lower population density and mobility.

Figure 1 Study population statistics. A: Gender distribution in the hepatitis B virus positive group, showing male predominance (male:female approximately 1.8:1); B: Distribution of patient by urban and rural areas; C: Clinical presentations of enrolled study patients; D: Prevalence of co-morbidities in the study population. Data represent the number of patients with specific co-existing medical conditions (e.g., hypertension, diabetes mellitus, chronic liver disease, cancer, chronic kidney disease, tuberculosis and liver damage/cirrhosis/transplant) at the time of enrollment.

The analysis of incidentally detected HBV patients on the basis of clinical presentation showed common complaints of abdominal pain, nausea, vomiting, fever, cough and diarrhoea (Figure 1C). The co-morbidities associated with these patients were hypertension, diabetes mellitus, chronic liver disease, cancer, chronic kidney diseases, tuberculosis, liver damage/cirrhosis/transplant (Figure 1D).

Haematological and biochemical comparisons

Comparative evaluation between the HBV-positive group and healthy controls revealed statistically significant differences in several haematological and liver function parameters (Figure 2). Haemoglobin levels were markedly reduced in HBV-positive individuals (P < 0.0001), suggesting anaemia or subclinical nutritional deficiencies. In contrast, neutrophil percentages were significantly elevated (P = 0.0006), while lymphocyte percentages were notably reduced (P = 0.0031), indicating possible immune dysregulation or chronic inflammatory response in HBV-infected individuals. Among the liver enzymes, SGOT and ALT were significantly increased in the HBV group (P = 0.0013 and P = 0.0001, respectively), consistent with hepatic inflammation or injury. Other parameters, including total leukocyte count, eosinophil and monocyte percentages, platelet count, total bilirubin, and SGPT, did not show statistically significant differences between the groups. Table 1 shows the comparison of significantly altered hematological and biochemical parameters between HBV-positive patients and healthy controls.

Figure 2 Comparison of heamatological parameters between the hepatitis B virus positive patients and healthy controls. HB: Heamoglobin; HBV: Hepatitis B virus; TLC: Total leukocyte count; SGOT: Serum glutamic-oxaloacetic transaminas; SGPT: Serum glutamic-pyruvic transaminase; ALT: Alanine aminotransferase.

Table 1 Comparison of significantly altered hematological and biochemical parameters between hepatitis B virus positive patients and healthy controls.

Parameters	HBV median (range)	Control median (range)
Hb (g/dL)	11.1 (5.3-17.9)	14.8 (12.4-17)
Total leucocytes (WBC) count/μL	8100 (2800-21000)	7300 (4900-9000)
Platelet count/μL	217000 (45000-543000)	264000 (167000-380000)
RBC count/μL	4 (2.14-5.94)	Not done
Neutrophils (%)	67 (32-96)	55 (42-68)
Eosinophils (%)	2 (0-9)	2 (1-5)
Basophils (%)	0	Not done
Lymphocytes (%)	23 (2-45)	32.5 (3-38)
Monocytes (%)	8 (1-16)	7 (3-10)
Bilirubin-total (mg/dL)	0.61 (0.16-11.55)	0.855 (0.26-1.59)
Bilirubin-conjugated (mg/dL)	0.285 (0.1-9.24)	Not done
Bilirubin-unconjugated (mg/dL)	0.34 (0.06-3.14)	Not done
SGOT (U/L)	28.5 (10-1237)	18 (15-36)
SGPT (U/L)	22 (9-1731)	16.5 (10-47)
Alk.Pho (U/L)	91 (46-337)	Not done
Protein (total, g/dL)	7 (3.9-8.9)	Not done
Albumin (g/dL)	3.8 (1.8-6.3)	Not done
Globulin (g/dL)	3.1 (1.7-5.3)	Not done
Albumin-globulin ratio	1.24 (0.46-2.05)	Not done

HBV: Hepatitis B virus; Hb: Heamoglobin; WBC: White blood cell; RBC: Red blood cell; SGOT: Serum glutamic-oxaloacetic transaminas; SGPT: Serum glutamic-pyruvic transaminase; Alk.Pho: Alkaline phosphatase.

HBV DNA viral load analysis

Out of the 100 HBsAg-positive individuals enrolled, 68 samples subjected to HBV DNA quantification. Among these, 17 individuals exhibited a viral load > 2000 IU/mL, 24 had < 2000 IU/mL, 11 were negative, 15 were invalid, and 1 was undetermined. The viral load distribution highlighted considerable heterogeneity in replication activity among the asymptomatic carriers. The lowest detectable viral load was 0.024 IU/mL, while the highest was 2033777.25 IU/mL.

Correlation analysis between biochemical markers and HBV DNA

A statistical correlation assessment was performed to evaluate the relationship between HBV DNA concentration and key liver function indicators. The analysis revealed a moderately strong positive association between viral DNA levels and serum SGOT (r = 0.55, P < 0.0003), SGPT (r = 0.43, P < 0.0065), conjugated bilirubin (r = 0.58, P < 0.01), and ALP (r = 0.37, P < 0.0232). These results indicate that elevated viral replication is linked to increased hepatocellular and biliary tract injury. In contrast, viral load demonstrated a negative association with serum albumin (r = -0.39, P < 0.014) and the albumin-to-globulin ratio (r = -0.35, P < 0.029), suggesting that higher viral burden may correspond to diminished hepatic synthetic capacity (Figure 3).

Figure 3 Correlation analysis between biochemical markers and hepatitis B virus viral load. A: Correlation between hepatitis B virus (HBV) viral load and serum glutamic-oxaloacetic transaminas; B: Correlation between HBV viral load and serum glutamic-pyruvic transaminase; C: Correlation between HBV viral load and alkaline phosphatase; D: Correlation between HBV viral load and albumin; E: Correlation between HBV viral load and albumin-globulin ratio. r was calculated using Graphpad prism V 8.0.1 software. SGOT: Serum glutamic-oxaloacetic transaminas; SGPT: Serum glutamic-pyruvic transaminase; ALP: Alkaline phosphatase.

DISCUSSION

The current study provides important insights into the haematological, biochemical, and virological characteristics of asymptomatic individuals diagnosed with HBsAg positivity, in comparison with healthy controls. Significant deviations were observed in their laboratory profiles, underscoring the subclinical impact of HBV infection.

The HBV cohort exhibited a mean age of around 40 years, reflecting earlier reports that middle-aged individuals in endemic areas are more frequently affected. A higher representation of males was noted, with a male-to-female ratio of approximately 1.8:1, which corresponds with epidemiological evidence indicating greater HBV occurrence and replication rates in men. These differences may be influenced by variations in immune responses linked to sex, as well as behavioural risk factors[21,22].

Significant alterations in haematological parameters, especially reduced haemoglobin levels and a shift in leukocyte profiles were evident in the HBV group. Anaemia, as indicated by significantly lower haemoglobin concentrations (P < 0.0001), may be attributed to chronic inflammation or mild bone marrow suppression associated with HBV. The increase in neutrophil percentages and decrease in lymphocyte counts may reflect an ongoing inflammatory response and immunological modulation, even in the absence of clinical symptoms. These findings support the hypothesis that HBV can induce low-grade immune activation in asymptomatic individuals[23].

Biochemical markers of liver injury were also significantly elevated in the HBV group. Increased serum transaminase levels (AST and ALT) suggest ongoing hepatocellular damage despite the asymptomatic presentation. This pattern is often observed in the immune-active phase of chronic HBV infection, which may progress silently over time. Interestingly, total bilirubin, albumin, and platelet counts did not differ significantly between groups, implying that while there is active liver injury, the hepatic synthetic and excretory functions remain largely preserved in this cohort. This is an important distinction, as it suggests that while the liver is being affected, overt decompensation may not yet have occurred[24,25].

Quantitative assessment of HBV DNA revealed wide inter-individual variability in viral loads. Approximately 25% of the tested individuals had viral loads above 2000 IU/mL, a threshold often considered clinically significant for determining treatment eligibility and monitoring disease progression. This heterogeneity highlights the dynamic nature of HBV replication, which may fluctuate over time and is influenced by host immune status, viral genotype, and other comorbid factors[26].

The correlation analysis further supports the relationship between viral replication and liver injury. There were notable positive correlations between HBV DNA levels and liver enzymes such as AST, ALT, and ALP, suggesting that higher viral loads are associated with greater hepatocellular and biliary injury. In contrast, negative correlations with serum albumin and albumin/globulin ratio indicate that increased viral activity may be associated with impaired protein synthesis or mild hepatic dysfunction, even before overt clinical symptoms manifest. These results align with earlier studies demonstrating that serum HBV DNA levels correlate with the degree of liver inflammation and fibrosis[27,28].

This study emphasizes the need for vigilant monitoring of asymptomatic HBV carriers. Despite the absence of clinical symptoms, a considerable proportion of these individuals exhibit laboratory evidence of hepatic inflammation and immune alterations. Early identification of those at risk for disease progression using combined biochemical and virological markers can inform timely clinical intervention and help prevent complications such as cirrhosis or hepatocellular carcinoma.

This study offers novel insight into HBV activity in asymptomatic individuals using hematological and biochemical parameters. The strength of the study lies in its well-characterized sample group and comprehensive laboratory-based evaluation, which allowed for detailed correlation between viral load and hepatic biomarkers. However, we also recognise limitation that the non-inclusion of hepatitis B envelope antigen testing could have provided additional clarity regarding viral replication status and infectivity. A subset of samples (invalid or undetermined HBV DNA results) could not be analyzed due to technical constraints, potentially limiting the granularity of subgroup comparisons. This is a small single centric study, further large multicentric cohort studies will be required to analyze.

CONCLUSION

This cross-sectional study highlights that asymptomatic individuals testing positive for HBsAg may still exhibit significant alterations in hematological and liver function parameters. Despite the absence of clinical symptoms, elevated levels of SGOT, ALT, and neutrophils, alongside decreased hemoglobin and lymphocyte counts, indicate an ongoing hepatic and immunological response. Moreover, wide variability in HBV DNA levels suggests differing degrees of viral replication, with a considerable subset of individuals exhibiting viral loads above the clinically significant threshold of 2000 IU/mL. Positive correlations between viral load and markers of hepatic injury, and negative correlations with albumin and albumin/globulin ratio, reinforce the impact of viral activity on liver function. These findings emphasize the need for routine laboratory monitoring in asymptomatic HBV carriers to enable early identification of disease progression. Early intervention through antiviral therapy or lifestyle modification may reduce long-term complications such as cirrhosis or hepatocellular carcinoma in this silent population. Further studies including large number of asymptomatic HBV positive carriers to be carried to provide deeper insights of virological profile.

ACKNOWLEDGEMENTS

We thank the patients participated in the study. The authors are grateful to Dr. D. Y. Patil Medical College, Hospital and Research Centre and Dr. D. Y. Patil Vidyapeeth, Pune, for providing necessary facility and support.