HEPATITIS B VIRUS IDENTIFICATION IN A BLOOD PLASMA AT A LOW VIRAL LOAD

Abstract

To analyze the method for detecting HBV DNA in peripheral blood at low viral load and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of blood and liver tissue biopsy material were used from 128 patients living in the Russian Federation and the Republic of Uzbekistan without CHB and with CHB confirmed detection of circle covalently closed HBV DNA in hepatocytes. Plasma viral load was measured using the «AmpliSens® HBV-Monitor-FL» kit. HBV at low viral load was detected by nested PCR. Analytical sensitivity was checked by step dilution. According to our method, at the first stage, an asymmetric PCR is carried out using extended oligonucleotide primers with different melting points, complementary to the hepatitis B different genotypes genomes greatest similarity region. To increase the sensitivity, a second PCR is performed using the first reaction amplification product and internal primers. The sensitivity of the method for DNA extraction from 100 μl of plasma was 5 IU / ml, specificity 100%. Since, in spite of the HBV genotypes characteristic geographical distribution, the detection of “alien” genovariants for certain territories is becoming more frequent, we tested the method in geographically remote but active international relations with the Russian Federation regions with a high frequency of hepatotropic viruses. The developed method for detecting HBV DNA in blood plasma at low viral load based on PCR technology allows the various HBV gene variants identification and genotyping, both characteristic and rare in the Russian Federation, circulating in other world regions . The method can be used to detect HBV in risk groups, in a population, as well as when screening blood donors in order to ensure the blood transfusions safety.