Does Hepatitis B Virus DNA Forecast Chronic Hepatitis in Hepatitis B Surface Antigen-positive Patients?

Abstract

Introduction:

Chronic hepatitis B (CHB) infection is accountable for nearly 240 million cases worldwide. Hepatitis B virus (HBV) has a propensity to show a divergent phase, and isolated measure of high HBV DNA level may not reflect the stage of the disease. In this background, this study was done to detect hepatitis B e antigen (HBeAg) and HBV DNA levels among hepatitis B surface antigen (HBsAg)-positive CHB cases and to determine the stage of disease with viral markers and biochemical parameters such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

Materials and Methods:

Blood samples were collected from 50 participants with CHB (HBsAg positive for more than 6 months) tested for HBe Ag by enzyme-linked immunosorbent assay, HBV DNA, ALT, and AST levels.

Results:

Among the 50 cases, 31 (62%) were HBeAg negative. Among these 31 HBeAg-negative groups, 16 (52%) had HBV DNA levels >2000 IU/mL. In these 16 patients, six had ALT >40IU/mL.

Conclusion:

HBsAg, HBeAg, ALT levels, and HBV DNA are important markers to determine the clinical stage of the disease. Since it is a chronic disease, patients need long-term follow-up. Further, emergence of viral mutants among vaccinated and those on lamivudine aggravates the existing problem.

INTRODUCTION

Hepatitis B virus (HBV) infection is the most extensive viral hepatitis posing public health problem globally. It is accountable for persistent infection in approximately 296 million cases and 820,000 deaths each year worldwide. It does not give rise to chronic infection all the time. If the infection is acquired during adulthood, only less than 5% of adults develop chronic hepatitis B (CHB) infection. Whereas infection in infancy or in early childhood approximately 90% develop CHB. Further, CHB progresses to cirrhosis, hepatocellular carcinoma, liver failure and death.^[1] Factors that influence the natural course of HBV infection include age, sex, immune status of the patient, viral load, replication of HBV, and other factors.^[2] Chronic hepatitis presents with necroinflammatory reaction in the liver with altered biochemical markers and clinical findings which persist for more than 6 months.^[3] The diagnosis of CHB has moved from serological to molecular methods. Since HBV has a propensity to show a divergent phase, isolated measure of high HBV DNA level may not reflect the stage of the disease. In this background, this study was done to detect hepatitis B e antigen (HBeAg) and HBV DNA levels among hepatitis B surface antigen (HBsAg)-positive CHB cases and to determine the clinical course of infection with viral markers and biochemical parameters such as alanine aminotransferase [ALT] and aspartate aminotransferase [AST] levels.

MATERIALS AND METHODS

Fifty CHB cases (HBsAg positive for more than 6 months) participated in this cross-sectional study. Five mL of blood was collected and serum was separated and stored at −80ºC. Samples were tested for HBeAg by enzyme-linked immunosorbent assay (ELISA) (Dia. Pro, Diagnostic Bioprobes S. r. l, Italy) and quantitative HBV DNA levels by real-time polymerase chain reaction (PCR) (Helini Biomolecules, Chennai).

The sensitivity and specificity of the ELISA kit is 100% (95% confidence interval: 99%–100%) and 97.5% (95% confidence interval: 1.2%–4.5%), respectively. As per the manufacturer’s instruction, positive and negative controls provided in the kit were included with the test run and the results were found satisfactory.

While performing HBV DNA PCR, controls (negative, positive, and internal controls) provided with the kit were used and the controls were satisfactory.

The Institutional Ethics Committee clearance (TIREC-Ref. No 1609/MICRO/2019) and written informed consent were obtained before starting the study. ALT and AST levels were collected from hospital records.

Inclusion criteria

Symptomatic or asymptomatic CHB patients aged above 15 years and HBsAg positive for more than 6 months.

Exclusion criteria

• Patients on antiviral treatment

• Concurrent infection with HIV or hepatitis C

• Renal/bone marrow recipients

• Hemodialysis patients

• Patients on chemotherapy

• Hepatic encephalopathy.

RESULTS

Of the 50 patients, 42 (84%) were between the ages 15 and 35 years. Thirty-seven (72%) were male and 13 (26%) were female. Two (4%) had a history of blood transfusion and 9 (18%) had a family history of hepatitis. Twenty-one (42%) had ALT >40IU/mL and 27 (54%) had AST >35 IU/mL.

Of the 50 patients, 31 (62%) were HBeAg negative. In these 31 HBeAg-negative patients, 16 (52%) had HBV DNA levels >2000 IU/mL and 5 (16%) had <2000 IU/mL. Among the 16 patients who were HBeAg negative with HBV DNA levels >2000 IU/mL, six had ALT >40IU/mL.

Among the 19 HBeAg-positive patients, 16 (84%) showed HBV DNA levels >2000 IU/mL and two (11%) had <2000 IU/mL [Table 1].

Table 1

Distribution of hepatitis B virus markers

Of the 16 cases (HBeAg positive with HBV DNA levels >2000 IU/mL), 12 had ALT >40IU/mL [Table 2]. This signifies a statistical association between ALT levels and HBV DNA levels.

Table 2

Alanine aminotransferase and aspartate aminotransferase distribution among the study population

DISCUSSION

The normal phenomena of HBV includes the immune tolerance stage (first stage) characterized by HBeAg, high levels of HBV DNA, but the ALT level will be normal and liver biopsy showed normal architecture. Next stage is the immune clearance phase (second stage) identified by positive HBeAg, fluctuating HBV DNA levels and consistent or periodic increase in serum ALT levels. Liver biopsy reveals active inflammatory changes in the hepatocytes. CHB inactive carriers (third stage) present with positive HBeAg or HBe antibody, normal ALT levels and low or absent HBV DNA in blood. Liver biopsy shows indistinct inflammation. Immune reactive CHB (fourth stage) is described as absence of HBeAg, presence of HBe antibody, HBV DNA and elevated ALT levels. Liver biopsy exhibits progressive inflammation. Occult CHB (fifth stage) is detected by absence of HBeAg, undetectable HBV DNA in the serum and normal ALT. Liver biopsy displays worsening inflammation.^[4]

The immune tolerant stage is common in those who acquire the infection from HBeAg-positive mother perinatally which may persist for couple of years to more than 30 years. After few years in this stage, many individuals develop to HBeAg-positive chronic hepatitis in the course of time, which may extend for several weeks to years. Then, the infection may go into inactive or develop into HBeAg-negative CHB after a quiescent stage. Patients are usually symptomatic during HBeAg-negative CHB infection, which is due to infection with precore or core promoter region mutant HBV variant preventing HBeAg production. Mutant HBV may evolve in chronic infection process, which may aid in immune evasion.^[5]

HBeAg-negative CHB is defined as:

HBsAg positivity and HBeAg negativity for more than 6 months, at least for a year. This yardstick eliminates patients with variable HBeAg seroconversion

Constant or occasional increase in ALT levels may be noted

HBV replication is being recorded by detection of HBV DNA in the serum and/or HBcAg in the liver.

If quantitative PCR (qPCR) is used, HBV DNA levels should surpass 10⁵copies/mL. Serum HBV DNA and/or HBcAg may not be measurable, especially during severe exacerbation of liver disease, and then serial testing is required to record the liver damage caused by HBV infection. Preceding to the raise of ALT level, serum HBV levels pump up remarkably. Almost 80% of individuals have high levels of anti-HBc IgM or total anti-HBc titers during or after ALT spikes. If these diagnostic yardsticks are not achieved, the liver cell damage in HBsAg-positive/HBeAg-negative cases may not be associated with replicating HBV but due to some other conditions.^[6]

Although HBeAg is an appropriate marker in the diagnosis and management of HBV infection, it cannot be a substitute for HBV DNA especially in HBeAg-negative CHB patients. It can be used as a supplementary test. HBV DNA by qPCR is a dependable and authentic test to diagnose and recognize the natural course of disease progression or regression. It also guides in monitoring therapy.^[7]

In the present study, among the 50 samples, 19 (38%) were HBeAg positive, while 31 (64%) were HBeAg negative. Similar results were recorded in the study by Rasika B et al, 23.3% of patients were found to be HBeAg positive, and 76.6% were HBeAg negative.^[7]

In this study, among the 16 HBeAg-negative cases with HBV DNA levels >2000 IU/mL, only six had ALT >40 IU/mL. Assessment of ALT or HBV DNA at one point is inadequate to nail down the ongoing disease status. Because, some HBeAg-negative CHB individuals can have persistently normal ALT (PNALT) over a period of time.^[8]

According to the European Association for the Study of Liver guidelines, PNALT is defined as “ALT <40 IU/L when measured every 3–4 months for 1 year.” Hence, serum ALT is tested every 3 months until 1 year before liver biopsy is indicated.^[9]

Liver biopsy is indicated in CHB individuals in the following situation, which is helpful to assess the course of the disease:

HBeAg positive, HBV DNA ≥20,000 IU/mL, mild or persistent or intermittent elevation of ALT, age above 40 years, no spontaneous seroconversion of HBeAg (3–6 months)

HBeAg negative, HBV DNA ≥2000 IU/mL, mild elevation of ALT, age above 40 years

HBeAg negative, HBV DNA ≥2000 IU/mL, persistently or intermittently elevated ALT, age above 40 years.^[3]

In this study, among the 31 HBeAg-negative cases, 16 (52%) showed HBV DNA levels >2000 IU/mL. Similar Akther et al stated that among the 48 HBe Ag negative cases, 29 (60%) had HBV DNA levels >2000 IU/mL.^[10]

CHB patients with HBV DNA levels >2000 IU/mL should be treated irrespective of their age due to the increased threat for HBV-related HCC as advocated by “The Gastroenterology Society of Australia.”^[11] Serum HBV DNA level more than 10,000 copies/mL is a substantial risk factor for HCC regardless of HBeAg status, level of ALT, and liver cirrhosis as recorded by Chen et al.^[12]

The patients with HBeAg negative and HBV DNA load <2000 IU/mL, without liver involvement, were screened for ALT levels every 3 months. As per the “National Viral Hepatitis Control Program”, antivirals will be initiated, if the ALT levels rise more than twice the upper limit.

This study emphasizes the value of definitive diagnosis of CHB among HBeAg-negative patients because management of these cases is discernibly distinct from inactive carrier state. Hence, quantitative estimation of HBV DNA is imperative in staging the progress of the disease.

As HBV is a vaccine preventable disease, vaccination should be made mandatory for all high risk population. Execution of scrupulous vaccination programs may decrease the prevalence of HBV, but this may also lead to an emergence of viral mutations which may pose a great challenge.

Limitations of the study

1. Liver biopsy to assess the course of the disease was not performed in the study participants.

CONCLUSION

HBsAg, HBeAg, ALT, and HBV DNA are important markers to determine the clinical stage of the disease. Further, emergence of viral mutants among vaccinated and those on lamivudine aggravates the existing problem. Since it is a chronic disease, patients should be monitored with viral and biochemical markers for a longer duration to prevent the catastrophes.