However, Axitinib 319460-85-0 HBV infection by sexual contact has recently become a prevailing alternative transmission route of HBV in Japan (30, 36). In particular, coinfection with HBV and human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, has been increasing among men who have sex with men (MSM), and the incidence of HBV infection associated with HIV-1-seropositive cases appeared to be 8.8%, which is higher than that in the general population (5). Thus, the epidemiology of HBV infection in Japan is quickly shifting. Here we report the most recent molecular epidemiologic status of HBV/HIV-1 coinfection. MATERIALS AND METHODS Sample. HIV/AIDS patients newly diagnosed at Nagoya Medical Center from 2003 to 2007 were tested for hepatitis B surface antigen (HBsAg), and HBsAg-positive patients were enrolled in the study.

Clinical data (age, gender, suspected route of HIV-1 infection, aspartate aminotransferase [AST] and alanine aminotransferase [ALT] plasma levels, CD4-positive T cell count, and HIV viral load) were obtained from medical records. Plasma HBV viral load was measured with COBAS TaqMan (Roche Diagnostics, Basel, Switzerland), and plasma HBc IgM titer was measured with Lumipulse (Fujirebio, Tokyo, Japan). The time of HBV infection was estimated by patient interview and HBc IgM titer results. This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Boards of the National Institute of Infectious Diseases and Nagoya Medical Center.

All patients provided written informed consent for collection of samples and subsequent analysis. Amplification of HBV and HIV DNA fragments and determination of DNA sequences. HBV nucleic acid was extracted from plasma using a MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics). As shown in Fig. 1, the full-length HBV genome was amplified in two fragments, L (3,167 bases) and S (624 bases). The primers used for amplifying HBV DNA were both newly designed and have been published previously (27). Details of these primers are summarized in Table 1. The DNA polymerases used for the first and nested PCRs were LA Taq (Takara, Shiga, Japan) and Prime Star HS (Takara) polymerase, respectively. The HBV genotypes were also determined using a commercial kit (Institute of Immunology, Tokyo, Japan) based on enzyme immunoassay to confirm that the results did not differ from those based on phylogenetic tree analysis. Entinostat Fig. 1. Genetic regions of HBV and HIV-1 used for phylogenetic tree analyses. The whole HBV genome was amplified in two fragments, L and S, and assembled. L and S fragments are indicated by single and double dashed lines, respectively. Table 1.