HDV infection was defined by positive anti-HDV results. Demographics and physical findings were recorded in all patients with or without selleck chem inhibitor HDV co-infection prior to the start of any anti-viral treatment. Biochemical and virological markers Serum samples of all patients were tested for biochemical parameters including complete blood count, total bilirubin, ALT, alkaline phosphatase, creatinine and prothrombin time (PT), by standard laboratory methods. Upper limit of ALT was 55 IU/L for men and 33 IU/L for women. Serological tests conducted were qualitative HBsAg, HB core IgG, HBeAg, anti-HBe, anti-HDV by EIA assay (Chicago, IL, USA) and anti-HCV by ELISA-3 in all included patients during their visits to clinic within six months.

All the biochemical, serologic and virological tests from the two centers was processed in the central clinical laboratory at the Aga Khan University Hospital, Karachi. HBV DNA PCR qualitative analysis was performed by Cobas Amplicor HBV Monitor (Roche Diagnostic Systems, Basel, Switzerland) with a lower detection limit of 500 copies/ml. We considered the value of the qualitative PCR detectable to be ��10 2. All these patients were subsequently tested for quantitative HBV DNA PCR assay (RoboGene HBV DNA Quantification Kit, TripleHyb version, Leipzig, Delitzscher Str, Germany) and anti-HDV testing. Quantitative HBV DNA PCR has the lower limit of detection of approximately 1000 (> 10 2) copies/ml. HDV RNA PCR qualitative assay (Roche Diagnostics, USA) could only be checked in 49 patients with HDV co-infection as this test was not available in early part of the study in our laboratory.

HDV RNA was isolated from patients’ serum samples by High Pure Viral RNA isolation kit, according to the manufacturer’s instructions (Roche Diagnostics, USA). RNA was eluted from spin columns provided with the kit in sterile nuclease free water and stored at -80 ��C until further analysis. Later, RNA samples were reverse transcribed into cDNA using 1st Strand cDNA Synthesis Kit for RT-PCR (Roche Diagnostics, USA). Briefly, cDNA mix consisted of reaction buffer containing 5 mM MgCl2, R NA, random primers, 50 units RNAse inhibitor and AMV reverse transcriptase. The reaction was carried out for 90 min at 42 ��C in a thermal cycler. The resulting cDNA was amplified with sequence specific primers for HDV. The amplified products were separated on a 1.5% agarose gel and a 400 bp product indicated the presence of HDV in the sample. To monitor the quality of the assay in each test Drug_discovery run, both negative and positive controls were included [20]. Abdominal ultrasound of all included patients was performed for the assessment of echo-texture, size and margins of liver & spleen and for features suggestive of portal hypertension.