This cross-sectional study was conducted in the Department of Microbiology at Shri MP Shah Government Medical College and Guru Gobind Singh Government Hospital in Jamnagar, Gujarat, India, over a period of one year, from September 2022 to August 2023. Approval for this study was obtained from the Institutional Ethical Committee (IEC) (169/04/2022), and informed consent was obtained from the patients. This was a time-bound study, and only the samples available during the study period were included.

Inclusion criteria: The patient samples from various indoor and outdoor departments of the hospital, sent for routine serological HBsAg investigations, received at the Microbiology department during the study period, were included in the study.

Exclusion criteria: Patients with HIV infection and dual infections with hepatitis A, C, and E were excluded from the study.

A total of 350 HBsAg positive samples were enrolled in the study. These samples were collected from a total of 30,000 samples received during the study period of one year. Approximately, 3-5 mL of venous blood was collected aseptically by venepuncture and transferred to a plain tube with a clot activator before being received in the laboratory. The samples were tested for HBsAg infection using the Merilisa HBsAg ELISA kit, a Direct Sandwich ELISA (Meril Diagnostic Pvt., Ltd., ICMR approved) [7]. Those who tested positive for HBsAg were subsequently tested for the detection of anti-HDV IgM antibodies using a capture ELISA kit [8]. The assay cut-off was calculated using the mean value of the Negative Control (NC) with the following formula: Cut-off = NC+0.250. Results were interpreted as a ratio of the sample’s Optical Density (OD) value to the cut-off value. Ratios for IgM antibodies <0.9 were considered negative, 0.9-1.1 were considered equivocal, and >1.1 were considered positive, as specified by the manufacturer (HDV IgM Dia.Pro Diagnostic Bioprobes Srl, ISO approved) [9]. Demographic and clinical findings such as age, gender, and risk factors- including blood transfusion and surgery- were recorded for all patients, as the transmission routes of HDV are similar to those of HBV [10]. Patients for whom no risk factors were identified were included in the “others” category. Subsequently, all data were analysed to determine the seroprevalence of HDV.

The statistical analysis was conducted using means and percentages. Differences between proportions were analysed using p-values calculated from Chi-square test. A p-value <0.05 was considered statistically significant.

Out of 350 HBsAg-positive patients, 10 (2.9%) were found to be positive for anti-HDV antibodies [Table/Fig-1]. So the seroprevalence of Hepatitis D among the HBsAg reactive patients was 2.9%.

Age-wise analysis in the present study showed a higher seropositivity among individuals in the age group of 51-60 years, followed by the age group of 21-30 years [Table/Fig-2].

Association with age and anti-HDV antibodies was not significant (p-value=0.08) [Table/Fig-3]. The gender-wise distribution of Hepatitis D antibody cases is shown in [Table/Fig-4].

There was no significant association between the presence of anti-HDV antibodies and gender of the cases in the study (p-value=0.07), as shown in [Tables/Fig-5], respectively.

The [Table/Fig-6] shows that the rate of positive cases was higher among individuals with a history of blood transfusion, which accounted for five cases (50%).

The HDV infection is associated with an increased risk of cirrhosis and hepatocellular carcinoma compared to those who only have chronic HBV infection [10]. Therefore, early diagnostic intervention in HDV-infected patients may be the right way to prevent end-stage liver disease and the development of cirrhosis.

Present study showed a prevalence of 2.9% of HDV antibodies among HBsAg-positive patients. This finding was consistent with similar studies conducted by Sonkar A et al., and Ajayi BB et al., which reported HDV seroprevalences of 2.1% and 3.3%, respectively [11,12]. Many factors can influence the prevalence of HDV infection, such as drug abuse, multiple sexual partners, HIV infection, geographical location, and a high incidence of HBV infection [13]. The seroprevalence of HDV was highest in the age group of 51-60 years, at 40%. In a study conducted by Tahaei SME et al., HDV seropositivity was observed in older patients [10]. Another study by Joseph K et al., also reported a high prevalence in older individuals [14]. The study revealed a female predominance over males. Present study finding was in agreement with the study carried out in Brazil by Lago BV et al., where only women were found to be HDV-infected, and the study by Gheorghe L et al., also showed a female predominance [15,16]. However, there was no association between HDV and variables such as age and sex, as the p-value was >0.05. In this study, hepatitis D antibodies were detected in 50% of cases with a history of blood transfusion, followed by surgery in 10%. According to a study conducted by Gheorghe L et al., showed 31% of a patients had a history of blood transfusion [16]. Another study by Shah L et al., also identified blood transfusion as the most common risk factor [17]. This could be attributed to the fact that multiple blood transfusions allow a large quantity of infective virions to enter a susceptible patient, increasing the chances of infection. However, there are differences in risk factors as well as lifestyle in different areas, so it is important to know the prevalence in specific regions.

HDV is the most severe form of chronic hepatitis. In resource-limited countries, it is challenging to conduct clinical trials, and consequently, HDV infection is endemic in these regions. However, due to significant advances in the characterisation of the viral life cycle, several host-targeting molecules are currently undergoing clinical evaluation with promising results. The seroprevalence of HDV infection was notably high in present study. Therefore, employing an HDV ELISA, which is a simple, easy, and less time-consuming procedure compared to molecular tests, can help screen patients for HDV infection. This approach may help reduce transmission and complications such as early hepatic decompensation, liver failure, and an increased risk of hepatocellular carcinoma compared to HBV monoinfection.

Virological replication markers such as HDV RNA and HBeAg were not measured for a more precise estimation of individual viral replication in the disease process due to cost constraints.

The seroprevalence of HDV was found to be 2.9% among HBsAg-positive patients in a study that predominantly involved middle-aged and older females. The most common route of transmission identified was blood transfusion. Co-infection with Hepatitis B aggravates the progression of liver diseases. Given that one of the primary routes of HDV transmission is blood transfusion, there is a clear need for blood screening for HDV, particularly in patients who have received multiple blood transfusions.