European Journal of Cardiovascular Medicine

Quantification DNA from the hepatitis B virus (HBV) is essential for treating persistent HBV infections. Chronic hepatitis B is a lifetime infection that can result from acute hepatitis B. Serious health issues, such as liver damage, cirrhosis, liver cancer, and even death, can result from untreated chronic hepatitis B. About 640,000 persons in the US are estimated by the CDC to have chronic (long-term) hepatitis B (Conners EE 2023). In 2022, non-Hispanic Asian and Pacific Islander individuals had the highest prevalence of chronic hepatitis B.(MK Weng, 2022). Hepatocellular carcinoma and HBV infection prevalence are epidemiologically related (Wong et al. 1993). Therefore, reducing HBV infection is a key public health objective in endemic areas.

The development of hepatocellular carcinoma is quite likely in cirrhotic patients (Lee WM. 1997).Despite a safe and effective vaccine, 50 million new cases are diagnosed annually. The incidence of HBsAg varies depending on a number of factors, including the country's health policy, the actions taken, education, and the state of the economy (Nebbia G et al. 2012). Sexual contact, close contacts, infected mother-to-newborn (perinatal, vertical), and parenteral contact with contaminated blood or bodily secretions (percutaneous) are the ways that HBV infects people.

 In serological approaches for diagnosing HBV, HBsAg is utilised as a screening test. The sensitivity of HBsAg screening tests is not always enough to identify HBV infection (Paraskevis et al.2010).In addition, HBsAg is utilised for serological diagnosis in conjunction with HBcAg, Anti-HBc IgM, Anti-HBc IgG, Anti-HBe, and Anti-HBs.One to two weeks following exposure, HBsAg, the primary indicator of HBV infection, can be detected; it removal is seen as an indication of viral clearance. The hepatitis B e antigen (HBeAg) signifies infectivity and represents active viral transcription and replication. Another indicator of continuous viral replication is serum HBV DNA: Importantly, higher HBV DNA levels are linked to worse outcomes and reflect higher quantities of the circulating virus. Molecular diagnostic methods have been developed in recent years. Particularly in situations when serologic approaches are insufficient and there is aberrant hepatitis B serology, molecular tools are being employed to identify mutant strains and clarify the mechanisms of HCC formation after antiviral therapy ( Premkumar M, Chawla YK. 2021) The nucleic acid sequence's in vitro propagation is one of the most crucial processes in molecular biology. Drug resistance, therapy monitoring, and illness identification might all be ascertained in this setting. As awareness of the significance of recognising hepatitis B virus DNA (HBV DNA) has grown, so too has the usage of these techniques. Real-time PCR is a quick and easy test that is commonly used to identify HBV DNA since it enables for the quantification of HBV DNA.

HBV is a small, circular DNA virus that is about 3.2 kb long. It has four genes with open reading frames (ORFs) that partially overlap. The polymerase protein, surface antigen, core antigen, and X protein are all encoded by these overlapping ORFs. Since HBV is extremely diverse and made up of closely related but distinct genomes, it is regarded as a viral quasispecies that exists within an infected person (Ngui SL et al. 1999). Up to 1011 virions are produced daily in infected individuals, demonstrating the rapidity of viral replication. 107 base-pairing errors can be produced daily over the 3200-bp genome due to the polymerase's high reverse transcription error rate (1 error/107 bases) during active infection (Locarnini S 2003).

The management of chronic hepatitis B has significantly improved due to recent developments in antiviral medication, which are based on the creation of new and more potent nucleos(t)ide analogues. This includes preventing allograft reinfection in patients receiving liver transplantation for HBV-related illness. The development of extremely sensitive diagnostics for tracking HBV DNA has contributed to the effectiveness of antiviral medication. The activity of HBV infection, the choice of patients for treatment, the effectiveness of antiviral medication, and the emergence of drug-resistant strains of HBV are all determined by molecular tests (Dandri M et al.2013, Kang SH et al.2017 ).The purpose of this study was to compare the HBV DNA results from our study's HBsAg-positive cases with those obtained from retrospective analysis of the real-time PCR method.

The current investigation was carried out at the Indian Council of Medical Research-DHR's Viral Research and Diagnosis Laboratory in Vijayawada, Andhra Pradesh. The objective in this study was to compare the results of HBV and HBsAg DNA in patients who tested positive for HBsAg. In this regard, serum samples from 128 patients who tested positive for HBsAg and were sent to our laboratory were reviewed after the fact. Following labelling, patient serum samples were kept at -80 degrees cold until analysis.

Quantitative PCR assay

Using a quantification standard (Amplicor HBV Monitor; Roche Molecular Diagnostics, Milan, Italy), the quantitative assay is based on competitive PCR. For DNA extraction and purification, 100 μl of serum was processed in accordance with the manufacturer's instructions. 50 microlitres of the purified DNA sample were mixed with 50 microlitres of the master mix that was ready for usage. The primer set utilised for the PCR amplifies a 104-bp fragment of the precore and core gene, and one of the primers was labelled with biotin. The target DNA and a quantitative standard (QS) were coamplified throughout the amplification process. A dinitrophenyl-labeled HBV-specific or QS-specific probe was used to hybridise the PCR products of the HBV core gene and the QS independently. The amount of HBV DNA in each specimen was determined by dividing the total HBV absorbance by the total HBV QS absorbance and the input number of HBV QS DNA molecules. The hybridisation products were identified colorimetrically. A standard curve created from each amplification run, with values ranging from 0 to 106 HBV copies/ml (1.7×105 IU/ml), was used to determine the number of copies per millilitre. The manufacturer states that the assay's detection limit is 300 copies/ml (52 IU/ml). The assay's dynamic range in the format we employed was 2×105 copies/ml (3.4×104 IU/ml) to 300 copies/ml (52 IU/ml).^.

Serum from 128 HBsAg-positive patients was tested for HBV DNA in our study. There were 35 (27.4%) females and 93 (72.6%) males. Five (4%) were children under the age of eighteen, and 123 (96%) were adults over the age of eighteen. The patients ranged in age from 11 to 80 years old, with a mean age of 37. Out of the 128 individuals in our study who tested positive for HBsAg, 102 (79.6%) had HBV DNA and 26 (20.4%) did not. The viral load in HBV DNA-positive cases was found to range from 174 copies/mL to 51,179 copies/mL. HBV DNA positivity was found in 27.4% of female patients and 72.6% of male patients (Figure 1).  Age-wise, HBV DNA was positive in 96% of 128 adult patients and 4% of 5 children with the disease (Figure 1).

Figure 1. HBV DNA positivity rates according to HbsAg, gender and age

HBV is prevalent both locally and globally. Due to liver tropism and its ability to produce hepatocellular carcinoma, it is one of the major hepatotrophic viruses. Public health is now concerned about it. According to the World Health Organisation, about 400 million people are chronic carriers of HBV, and approximately 2 billion people have been exposed to the virus. Fifty million new instances are added to these patients each year. Death results from both acute and chronic consequences, including cirrhosis and hepatocellular carcinoma brought on by HBV (Mıstık R 2007, Han SH, and Tran TT 2015).Since antiviral medication has significantly improved the prognosis of HBV disease in active carriers, measuring HBV DNA has emerged as the most straightforward and trustworthy technique for the precise management of chronic hepatitis B. Furthermore, hepatocellular cancer associated with HBV is significantly predicted by the level of HBV DNA (Chen, C. J et al.2006, Pollicino T et al. 2007). To monitor the viral load in serum and plasma, evaluate the effectiveness of treatment, and track the emergence of drug-resistant strains of HBV, highly sensitive and repeatable molecular assays are needed.           

Out of the 128 HBsAg-positive patients in this investigation, 35 were female and 93 were male. 102 samples had an HBV DNA viral load found in them. 31 (39.4%) of the patients were female, and 71 (69.6%) of the patients were male, when the viral load and DNA copies were found. According to research, men are more likely than women to be infected with the hepatitis B virus and have a 20–25% risk of doing so (Vojnosanit Pregl et al. 2023). Males are thought to have a higher risk of developing hepatocellular carcinoma (HCC) worldwide (Mayaphi SH et al. 2012). This high may result from a number of circumstances.Ijaz T. et al. found 68% of male patients and 32% of female patients in their study when the rate of HBsAg positivity was analysed by patient gender; in Turkey, Bati et al. found 63.5% of male patients and 36.5% of female patients. In line with previous research, we also discovered that males had higher levels of HBV DNA and HBsAg seropositivity than females. Males accounted for 72.6% of HBsAg-positive cases in our investigation, while 69.6% of HBV DNA copies were found in males. According to studies, men are more likely than women to be in public settings, which results in higher levels of HBsAg seropositivity in men (Sabeena et al. 2021). The prevalence of HBV infection is higher in men than in women because men are more likely to be exposed to potential risk variables like medication injections, barbershops for shaving, and sexual activity, all of which increase the chance of contracting HBV (Ijaz T et al., 2023).

According to this study, the age group with the highest incidence of hepatitis B contamination is 20 to 40 years old (77, 62.6%), followed by 41 to 80 years old (47, 36.4%). The age group with the lowest incidence rate was 5 to 19 years old (4%). People between the ages of 20 and 45 are more susceptible to the risk factors associated with HBV infection, taking into account the pathways of HBV transmission (Anjali H et al. 2012). Our results are consistent with those of Yewande et al. (2018), who also observe that a similar age range has an elevated incidence of HBV infection and that individuals over 45 and children younger than 15 exhibit less activities that increase their risk of contracting the virus (Obiomah C et al.2020). 4% of HBV-positive cases in our investigation involved individuals younger than 18. Sharing a toothbrush, chewing gum, or towel makes it easier for children under six to contract HBV. As a result, routine childhood Hepatitis B immunisation is crucial for preventing HBV infection and reducing carriers. Due to the rise in chronic liver disease brought on by early infection, vaccination has become increasingly crucial. 

The viral loads in HBV DNA-positive subjects in our investigation ranged from 174 to 51,179 copies/mL. Antiviral treatment is not recommended for every patient with hepatitis B infection, which has a wide range of virological and clinical manifestations. EASL 2017 guidelines state that patients with HBV DNA > 2000 IU/mL with at least mild fibrosis may begin treatment, while those with HBV DNA ≥ 2000 IU/mL should have ALT tests performed at least every three months for the first year and every six months for the next three years (Lampertico P et al.2017). We separated the patients in our study into two groups: those with HBV DNA levels below 2000 IU/mL (84%) and those with levels above 2000 IU/mL (16%). HBV DNA levels above 2000 IU/mL, for instance, were linked to a significantly higher risk of HCC and mortality than lower viral loads, based on numerous seminal long-term cohort studies (Madihi S et al.2020).

The current study concludes by demonstrating an automated, labor-saving platform for measuring HBV DNA in serum that enables quick, precise, and highly sensitive measurement of HBV DNA levels throughout a broad dynamic range.It precisely measures the amount of HBV DNA present in samples taken from individuals who have a persistent HBV infection. Over the whole dynamic range of quantification, which includes values seen in both treated and untreated patients with chronic hepatitis B, quantification is linear. In the future, researchers will need to analyse samples with HBV genotypes A through F and obtain information on irregular nucleotide variations in genotypes.The management of both acute and chronic HBV infections, the evaluation of antiviral medication, and the detection of drug resistance may all benefit from the system.

Acknowledgement

The authors sincerely thank the Secretary to Government of India, Department of Health Research and Director General, Indian Council of Medical Research for financial support under the State VRDL Project (Ref.No.DHR/VDL/35/2015). The authors thank Dr. K.Sudhakar, Principal, Siddartha Medical College,Vijayawada for his kind support and encouragement during the study. We also thank the technical support and staff of VRDL.

The present study evaluated the effectiveness of implementing Self-Directed Learning (SDL) among Phase I MBBS students in Biochemistry through two approaches—Traditional Lecture followed by SDL and SDL-only sessions. Both approaches resulted in significant improvement in post-test scores, confirming the value of SDL in enhancing knowledge acquisition and retention. However, the Traditional Lecture + SDL group demonstrated significantly higher post-test performance (14.2 ± 2.1) compared to the SDL-only group (12.5 ± 2.3; p = 0.02), highlighting the importance of structured input before independent learning in novice learners.

Student perceptions revealed that SDL promoted active participation, deeper conceptual understanding, and confidence in self-learning. Students expressed that combining faculty-guided lectures with SDL provided clarity and direction, which was particularly valuable at the beginner stage.

Faculty perspectives aligned with these findings, emphasizing that while SDL fosters lifelong learning skills, Phase I students require scaffolding and guided facilitation to optimize learning outcomes. Faculty endorsed a blended approach, noting that it improves student engagement, critical thinking, and accountability while gradually transitioning them toward independent learning.

Limitations of the Study: This study was conducted in a single institution limiting generalizability, focussed solely on cognitive domain without assessing skills or attitudes. Time-bound SDL sessions may not reflect the extended learning cycles required for true self-directed learning.

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