Study design and population
This was a descriptive cross-sectional, laboratory based study conducted at National Blood Transfusion Service (NBTS) in Asmara, which is the capital city of Eritrea and serves about 20 referral hospitals [20]. It supplies blood and blood products and serves hospitals from six administrative regions (Zobas), including Maekel, Debub, Anseba, Gash-Barka, Semienawi Keiyh Bahri and Debubawi Keiyh Bahri.
A total of 23,232 infected blood donors were screened for serological status of HBV, Hepatitis C Virus (HCV), Human Immunodeficiency virus (HIV) and syphilis (Treponema pallidum) between January 2015 and December 2017 as part of routine blood donation requirement. Of 395 blood donors tested positive for HBsAg. Considering availability of resources 191 samples fulfilling a pre-set criteria (aged between 16 and 65 years and a body weight greater than 50 kg), were randomly selected for inclusion in the study. HBsAg serum reactive samples were labelled with unique identifier code and stored at -20˚C until further investigation.
Serological assays
Enzyme Linked Immuno-Sorbent Assay [21] kits were used to screen for HBsAg (SD Standard Diagnostic, Republic of Korea), anti-HCV (SD Standard Diagnostic, Republic of Korea) and anti-T. pallidum (MEDDIFF, France). While HIV screening was done using an AccuDiag™ HIV 1&2 Ag/Ab (Diagnostic Automation/Cortez Diagnostic, California, USA). HBV-positive serum samples (n = 191) were re-tested for HBcAb. Those with detectable HBcAb were further tested for hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (HBsAb) using ELISA kits (Fortress Diagnostics, UK). Only those with HBcAb-total and HBsAg were then processed for DNA extraction and screened for other HBV serological markers, including HBeAg and HBeAb using ELISA kits (Fortress Diagnostics, UK).
HBV DNA extraction
HBV DNA was extracted from 200 μl of serum using an Analytik Jena DNA mini-extraction kit (Analytik Jena, Germany) according to the manufacturer’s instructions. The extracted DNA was then eluted in 60 µl of pre-heat RNase-free water before storing at -20˚C until use.
Amplification of HBV S-gene for genotyping
A genotyping system based on multiplex-nested PCR with type-specific primers (Macrogen company, Seoul, Korea) was used. This process was undertaken for HBV genotypes A to F. Based on pre-S1 region in S genes of the HBV genome were targeted as described previously by Naito et al. (2001) [22]. The first-round PCR was performed using two universal outer primer pairs: P1 (sense) - 5'-TCACCATATTCTTGGGAACAAGA-3') and S1-2 (antisense) - 5’CGAACCACTGAACAAATGGC-3’. For second-round PCR, B2 was used as the inner primer (sense) (5’ - GGCTCMAGTTCMGGAACAGT-3’), with a combination of antisense primers called mix A for genotypes A, B and C. The antisense primers used were: BA1R (genotype A specific, 68 bp) (5’-CTCGCGGAGATTGACGAGATGT-3’); BB1R (genotype B specific, 281bp) (5’-CAGGTTGGTGAGTGACTGGAGA-3’); BC1R (genotype C specific, 122bp) (5’-GGTCCTAGGAATCCTGATGTTG-3’). While B2R was used as the inner primer (antisense) (5’-GGAGGCGGATYTGCTGGCAA-3’), with a combination of antisense primers called mix B for genotypes D, E and F. The antisense primers used were: BD1 (genotype D specific, 119 bp) (5’-GCCAACAAGGTAGGAGCT-3’); BE1 (genotype E specific, 167 bp) (5’-CACCAGAAATCCAGATTGGGACCA-3’); BF1(genotype F specific, 97bp) (5’-GYTACGGTCCAGGGTTACCA-3’).
The total volume of the reaction mixture was 20 µl. The mixture contained 16 µl of nuclease-free water, 1 µl (10 pmol) each of P1 and S1-2 primers and 2 µl of DNA sample, all of which were added to the tube of the ready mix containing 2.5 mM dNTP mix, 2.5 U Taq DNA Polymerase, 1x reaction buffer, and 1x gel loading buffer (iNtRON, Biotechnology, Korea). The mixture was centrifuged at 5,000 rpm for 15 seconds. The master cycler gradient (Eppendorf, Germany) was programmed as follows: incubation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and elongation at 72°C for 2 min. The final elongation step was performed at 72°C for 5 min.
Two second-round PCRs were performed for each first-round PCR product using either
B2 and mix A group for genotypes A to C or B2R and mix B group for genotypes D to F. Firstly, 3-ml aliquot of the first PCR product was added to mix A and mix B. The reaction mixture of mix A or B (iNtRON, Biotechnology, Korea) contained 15 µl of nuclease-free water and 2 µl (10 pmol) of respective primers. Amplification conditions composed of 40 cycles of preheating at 95°C for 5 min, followed by 20 cycles of amplification at 95°C for 20 s, 60°C for 20 s and 72°C for 30 s and an additional 20 cycles of 95°C for 20 s, 62°C for 20 s and 72°C for 30 s.
Detection of HBV Genotypes
HBV genotypes for each serum sample were determined by identifying genotype-specific DNA bands. Two different second-round PCR products from one sample were separately electrophoresed for 1 h and 10 min at 100 V in 1.5% agarose gel D-1Low EEO (Eppendorf, Italy) stained with gel red (Biotium, USA). The gel was then observed and evaluated in UV light transilluminator (UVP, UK). The size of PCR products were estimated by comparing them to the migration pattern of a 50-bp DNA ladder (iNtRON, Biotechnology, Korea).
Data collection and analysis
The data of HBV infected blood donors were retrieved from the NBTS Delphyn Blood Bank Data Management version 7.6.10.0 database and transferred to Excel Spreadsheet (Microsoft Inc.). Meanwhile, the records of specific demographic information of all blood donors (age, sex, occupation, marital status, educational status, regions (Zobas) and blood group types (e.g., ABO and Rh) were retrieved with the assistance from the authorised staff of blood bank data center. Statistical analysis was analysed using the SPSS version 25.0 (IBM Corporation, Chicago, IL, USA). Basic statistics, such as percentages and mean ± standard deviations (SD) were used to summarise the data, where appropriate. The baseline characteristics of blood donors were analysed using the Chi-square (χ2) test or Fishers exact test (categorical variables). A two-tailed p-value less than 0.05 was considered statistically significant.