Backgrund and aim: Detection of HBV-DNA in HBsAg negative patients is defined as occult HBV infection. Previously, we investigated the prevalence and the genomic heterogeneity of occult HBV in the liver of 24 HIV+/HbsAg negative patients: 8 HBsAg- patients were HBV-DNA positive (33%) in the liver. HBV can be detected in PBMCs, so infected PBMCs can act as reservoirs for the cell-to-cell transmission of the virus. We aimed to determine the prevalence of occult HBV infection of peripheral blood mononuclear cells, defined as detection of sequences from ≥2 HBV genes in subjects HIV+ with HBV occult infection in the liver and to detect the viremia of HBV both in the liver than in the PBMCs. Methods: We focused on 6 HIV+ patients with occult HBV in the liver: HBV DNA levels were quantified using a real-time PCR assay based on Light Cycler technology revealed through SYBR green fluorochrome both in the liver than in PBMCs. Nested-PCR and direct sequencing were performed for S, X and PreC/Core regions. The controls were 14 HIV/HBV coinfected patients: liver+PBMCs+serum of 5 patients and serum+PBMCs of 9 patients. Results: five individuals positive for anti-HBc but negative for HBsAg had HBV DNA in their PBMCs. These 5 were also positive for HBV genomes in their liver but not in their serum. HBV-DNA, indeed, was detectable in liver, PBMCs and serum of all controls. SYBR green real-time RT-PCR showed a high level of sensitivity as the detection limit of technique was assessed at 20 HBV-DNA copies/15ng liver DNA and 20 HBV-DNA copies/300 ng DNA/10 mln cells.We found a low viremia of HBV in liver of all patients with occult infection . So in the PBMCs: 2 cases were <20 HBV-DNA copies/300 ng DNA/10 mln cells Conclusions: This study confirm previous data showing that detection of viral genomes in PBMCs may occur in the absence of viremia, antigenemia or specific viral antibodies in serum. Moreover, in the clinical setting, the most difficult parts of HBV-DNA research include the requirement of liver biopsies, and the lack of sensitive, specific and quantitative methods to detect HBV-DNA from biopsies of HBsAg negative patients. We found that the real time PCR teqnique can be applied for detection of copies of HBV in the liver and PBMCs of patients with HBV occult infection
R. Cassini, M.S. De Mitri, D. Gibellini, G. Lettini, I. Santeramo, G. Verucchi (2010). Occult HBV infection in PBMCs of HIV+ individuals.
Occult HBV infection in PBMCs of HIV+ individuals
CASSINI, ROMINA;DE MITRI, MARIA STELLA;GIBELLINI, DAVIDE;VERUCCHI, GABRIELLA
2010
Abstract
Backgrund and aim: Detection of HBV-DNA in HBsAg negative patients is defined as occult HBV infection. Previously, we investigated the prevalence and the genomic heterogeneity of occult HBV in the liver of 24 HIV+/HbsAg negative patients: 8 HBsAg- patients were HBV-DNA positive (33%) in the liver. HBV can be detected in PBMCs, so infected PBMCs can act as reservoirs for the cell-to-cell transmission of the virus. We aimed to determine the prevalence of occult HBV infection of peripheral blood mononuclear cells, defined as detection of sequences from ≥2 HBV genes in subjects HIV+ with HBV occult infection in the liver and to detect the viremia of HBV both in the liver than in the PBMCs. Methods: We focused on 6 HIV+ patients with occult HBV in the liver: HBV DNA levels were quantified using a real-time PCR assay based on Light Cycler technology revealed through SYBR green fluorochrome both in the liver than in PBMCs. Nested-PCR and direct sequencing were performed for S, X and PreC/Core regions. The controls were 14 HIV/HBV coinfected patients: liver+PBMCs+serum of 5 patients and serum+PBMCs of 9 patients. Results: five individuals positive for anti-HBc but negative for HBsAg had HBV DNA in their PBMCs. These 5 were also positive for HBV genomes in their liver but not in their serum. HBV-DNA, indeed, was detectable in liver, PBMCs and serum of all controls. SYBR green real-time RT-PCR showed a high level of sensitivity as the detection limit of technique was assessed at 20 HBV-DNA copies/15ng liver DNA and 20 HBV-DNA copies/300 ng DNA/10 mln cells.We found a low viremia of HBV in liver of all patients with occult infection . So in the PBMCs: 2 cases were <20 HBV-DNA copies/300 ng DNA/10 mln cells Conclusions: This study confirm previous data showing that detection of viral genomes in PBMCs may occur in the absence of viremia, antigenemia or specific viral antibodies in serum. Moreover, in the clinical setting, the most difficult parts of HBV-DNA research include the requirement of liver biopsies, and the lack of sensitive, specific and quantitative methods to detect HBV-DNA from biopsies of HBsAg negative patients. We found that the real time PCR teqnique can be applied for detection of copies of HBV in the liver and PBMCs of patients with HBV occult infectionI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
