*2.1. Setting*

Bahrain is a small archipelago country (33 islands) that is situated near the western shores of the Arabian Gulf with a total area of 765.3 km2. It has a total population of 1,234,571, out of which 666,172 are non-Bahraini and it is one of the most densely populated countries in the world (1461/km2) [18].

#### *2.2. Study Design*

In this cross-sectional study, patients with established chronic hepatitis B infection (positive for HBsAg antigen, HBcAg antibody) referred to Bahrain Defense Force Hospital (second largest hospital in Bahrain) were investigated for HBV genotypes. The major inclusion criterion was testing positive for HBsAg for over six months (chronic infection), with levels of ALT around the normal range. All patients were negative for antibodies against hepatitis C and human immunodeficiency virus.

#### *2.3. Sample Collection, HBV DNA Extraction and Genotyping*

Five ml of venous blood were collected from patients and sera were separated and stored at −80 ◦C prior to HBV genotyping. HBV DNA extraction, PCR amplification of DNA, and genotyping were carried out according to the instruction while using a commercially available kit (SMITEST, MBL Co. Nogoya, Japan) based on hybridization with type-specific probes immobilized on a solid-phase support. The quality and quantity of extracted DNA was determined with a Nano drop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) after DNA extraction. Serological investigation for HBsAg and HBeAg were carried out while using commercial enzyme-linked immunosorbent assay (ELISA) kits (ARCHITECT ANALYSER i2000, Abbott, Santa Clara, CA, USA). Liver function tests; Total and Direct Bilirubin, Alanine Transaminase (ALT), Alkaline Phosphatase (ALP), Aspartate Transaminase (AST), Gama Glutamyl Transferase ( γGT) and Lactate Dehydrogenase (LDH) were analyzed in serum samples while using routine biochemistry analyzer (COBAS c 501, Roche/ Mannheim, Germany).

#### *2.4. Statistical Analysis*

Statistical analyses were performed while using IBM SPSS Statistics 23 software. For baseline variables, summary statistics employed frequencies data, mean, and standard deviation (SD) for continuous variables. Continuous variables were compared using unpaired t-tests and ANOVA as appropriate. Descriptive Statistics were calculated to determine the prevalence of genotype with respect to data of demographic factors, serology, and other investigations. Univariate analysis of Variance (ANOVA) was applied to determine any significance di fferences for the genotype and liver function tests.

## *2.5. Ethical Approval*

The local ethics and research committees of Bahrain Defense Force Hospital and University of Bahrain approved this study (approval date 7 September 2017, Project code: 2017/12). It conformed to the provisions of the Declaration of Helsinki in 1964 (and revised in Fortaleza, Brazil, October 2013). All of the patients signed the informed consent form before participation.
