Study Site and study population
A cross sectional survey was conducted in 18 townships of all States and Regions of Myanmar from May to October, 2015. Eighteen townships were selected from 7 States (Kachin, Kayah, Kayin, Chin, Mon, Shan and Rakhine), 7 Regions (Bago, Sagaing, Magway, Ayeyarwady, Tanintharyi, Yangon and Mandalay) and Nay Pyi Taw Union Territory. A total of 5,547 subjects (aged between 15 to 80 years, both sexes) were participated in the survey.
Sampling procedure and recruitment
Two-stage cluster sampling method was used and selection of primary sampling units (PSUs) was performed that one township was randomly selected, which is considered to have average level of viral hepatitis B infection in all States and Regions of Myanmar. Selection of secondary sampling units (SSUs) was performed that from each selected PSU (township), 10 wards and villages were selected according to probability to population size. From each selected SSU (ward/village), 30 households were selected using systematic random sampling. The sampling frame for this sampling is the list of households available from the Basic Health staff. One eligible participant aged between 15 to 80 years in the selected households was recruited by random sampling.
Screening of HBV infection and sample collection for genotyping study
HBV screening was carried out at the field sites using SD Bioline HBsAg WB (Cat. No 01FK10W, Standard Diagnostic, Inc., Korea) according to manufacturer’s instruction. HBs Ag positive subjects were invited for counseling about consequences of infections, treatment options and further testing for HBV genotyping. The field investigators explained the purpose and procedure of the study and informed consent was obtained from each subject before the 2 milliliter venous blood samples were taken. Sera were separated and transported to the Department of Medical Research for further genotyping testing. A total of 353 HBsAg positive subjects, 147 males and 206 females with the mean age of 35.5 years (SD=10.8) were included in this genotyping study.
Confirmation of HBs Ag positive serum samples
HBs Ag positive serum samples by ICT (Immuno-chromatographic Test) were further confirmed by commercially available immunoassay kit, HBs Ag ELISA 3.0 assay (Cat. No 01EK10, Standard Diagnostic Test Kit, SD, Korea). The tests were performed according to the manufacturer’s instruction.
Viral DNA extraction
The ELISA confirmed HBs Ag positive were undergone viral DNA extraction which was performed with the QIAamp DNA Mini kit (Qiagen, Inc., Hilden, Germany), according to the manufacturer’s instructions.
Amplification of pre-S gene of HBV by PCR
The HBV PreS gene was amplified with nested PCR using PF-PR and NF-NR primers sets (PF 5' TTG GAC TCA CAA GGT GGG AA3'; PR 5' GTC CAC CAC GAG TCT AGA CTCT 3'; NF-5' TCA TTT TGT GGG TCA CCA TAT 3'; NR-5' CTG TAA CAC GAG CAG GGG T3'). Five microliter (μl) of extracted HBV DNA was amplified in a PCR mix including Tris HCL buffer, 2 mM Magnesium Chloride, 0.1 mM dNTPs, 2 units taq polymerase (Cosmo) and 0.25 μM each of the primers. The PCR thermal cycling profile was; 5 minutes at 94ºC then 30 cycles including 30 sec at 94ºC, 30 sec at 51ºC and, 45 sec at 72ºC, and then 10 min at 72ºC. Negative samples after first round PCR were amplified in nested PCR using second round primer set and a thermal profile like the first round but repeated for 35 cycles instead of 30 with annealing 54ºC. After confirming by gel electrophoresis, the products were purified with SV column PCR purification kit (GeneAll Biotech, Korea) according to the manufacturer’s instruction.
Determination of HBV Genotypes by direct sequencing of preS gene
The purified PCR products were subjected to sequencing by chain termination method using commercially available Kit (Big Dye Terminator Cycle Sequencing Kit, Applied Biosystems). Briefly, 2 μl of purified DNA was mixed with 1.85 ul of 5x sequencing buffer, 0.25 μl of Big dye terminator, 0.5μl of 0.125 μM primer (Forward or Reverse) and 5.4μl of water. The thermal profile used was; 35 cycles of 60 sec at 96ºC; 05 sec at 50ºC and 3 min at 60ºC. The 3500 XL Genetic Analyzer (Applied Biosystems) was used for Sanger sequencing method .
Determination of HBV genotypes and sub-genotypes
HBV genotypes were determined using the sequences of pre-S/S genes with NCBI Web based HBV Genotyping Tool (http://www.ncbi.nlm.nih.gov/projects/genotyping/ formpage.cgi). HBV DNA sequences were aligned with reference sequences using CLUSTAL method (MedAlign, Lasergene, DNASTAR Inc., Madison, WI) and manually edited the sequences with BioEdit Sequence Editor (version 7.2.5) and phylogenetic relationships were estimated by neighbor- joining method . For determination of sub-genotype, the study sequences were aligned with published sequences representing all known HBV sub-genotypes. Multiple sequence alignment was performed using the built-in ClustalW integrated in MEGA 4.20 . HBV sub-genotypes were determined by phylogenetic analysis in MegAlign software using the neighbour-joining method with a bootstrap test. The nucleotide sequences in this study have been deposited in the NCBI Gene Bank database. (Accession number: MH816993-995 and MH925817-925683)
All statistical analysis was performed using with Statistical Program for Social Science Software (SPSS) 23.0 software for Windows (SPSS Inc., Chigago IL., USA). Comparison between categorical variables was tested by chi-square test. A P-value (two tailed) of less than 0.05 was considered to be statistically significant. In this genotyping study, distribution of HBV genotypes were compared among 5 geographical areas as central, east, north, south and western part of Myanmar. Mandalay, Magway, Nay Pyi Taw, Bago, Ayeyarwady and Yangon regions were collectively described as central area , Shan and Kayah states as eastern area, Kachin state and Sagaing regions as northern area and Mon and Kayin states, Tanintharyi region as southern area and Rakhine and Chin states as western area. HBV sub-genotypes results were analyzed collectively.