Prevalence of hepatitis delta virus infection among hepatitis b virus surface antigen positive patients circulating in the largest province of pakistan

A total of 200 sera samples that were found positive for HBV by rapid Immunoblot screening methods were received from 2009 to 2010 that were included in this study. A filled standard form containing subject's demographics and risk behaviors were also received along with each sample. Out of these, 96 samples were found positive for hepatitis B surface antigen (HBsAg) by Enzyme linked Immunosorbant Assay (ELISA) method and were used for anti-HDV ELISA analysis. Out of these, 80 anti-HDV ELISA positive samples were further analyzed by HDV PCR. Total 56 anti-HDV ELISA positive samples were found negative for HDV RNA by PCR. All the samples belonged to different regions of Punjab, Pakistan. A filled standard form containing subject's demographics and risk behaviours were also received along with each sample.

Initially all the patient's sera were checked for HBsAg using an ELISA assay kit (DRG Instruments, Germany). Anti-HDV antibody was detected in using 3^rd generation Enzyme ELISA (DIA.PRO, Diagnostic Bioprobes Srl Italy) kits using the methodology described in the manufacturer's protocol.

HDV RNA was extracted from 100 μL serum samples using Gentra (Puregene, Minneapolis, MN 55441 USA) RNA Isolation kit according to the manufacturer's protocol. The extracted RNA (10 μL) was reverse transcribed into cDNA with Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Life Technologies Inc., USA). Briefly, 1 μl of HDV-specific antisense primer (10 pmol/μl) nucleotides 873-896 {5'-CCGCGAGGAGGTGGAGATGCCATG-3'} was mixed with 10 μL of extracted RNA followed by incubation at 70°C in thermal cycler for 10 minutes. Then cooled on ice for 2 minutes and added 9 μL of RT Mix that contained 50 mM Tris-HCl (pH 8.3), 7.5 mM KCl, 3 mM MgCl₂, 0.1 M DTT, 10 mM dNTPs and 200 U of MMLV reverse transcriptase enzyme. cDNA was synthesized at 37°C for 50 minutes and then heat inactivated the MMLV Enzyme at 95°C for 3 minutes. Spun down and stored at -20°C till was used in nested PCR.

The qualitative detection of HDV-cDNA was carried out by nested PCR using four μl of synthesized HDV-cDNA. The first round PCR was done in a tube containing 20 μl of PCR reaction mixture (2.5 mM MgCl2, a 100 μM concentration of each of the four deoxynucleotides (dNTPs), 10 pM of each outer sense nucleotides 695-718 {5'-CATGGTCCCAGCCTCCTCGCTGGC-3'} and outer antisense primers nucleotides 873-896 {5'-CCGCGAGGAGGTGGAGATGCCATG-3'} [11] and 1 U of Taq DNA polymerase Enzyme). The thermocycler (ABI PCR system 2700; PE Applied Biosystem Inc., USA) was programmed to initially incubate the samples for 2 min at 95°C, followed by 35 cycles consisting of 95°C for 1 min, 64°C for 1 min, and 72°C for 1 min. Second round PCR was performed with the same reaction mix and amplification conditions using inner sense nucleotides 729-748 (5'-CAACATTCCGAGGGGACCGT-3') and inner antisense primers nucleotides 846-865 (5'-GAAGGAAGGCCCTCGAGAACAAGA-3') [11]. Standard precautions to avoid contamination during PCR were taken. Positive controls (HDV ELISA & PCR positive) and negative controls (negative HDV serum and distilled water) were included in each run. Finally the PCR products were electrophoresed on a 2% agarose gel prepared in 1× Tris-borate-EDTA (TBE) buffer, stained with ethidium bromide, and evaluated under UV transilluminator. The sizes of PCR products were estimated according to the migration pattern of a 50-bp DNA ladder (Fermentas Life Sciences). The sizes of the first round PCR products and nested PCR were 202-base pair (bp) and 137-bp respectively.