High Prevalence of Hepatitis B Virus Markers in Romanian Adolescents With Human Immunodeficiency Virus Infection

Simona Maria Ruta, MD, PhD; Rodica Floarea Matusa, MD, PhD; Camelia Sultana, MD; Loredana Manolescu, MD; Claudia A. Kozinetz, PhD; Mark W. Kline, MD; Costin Cernescu, MD, PhD

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Patients and Methods

Demographic, social, and clinical data were obtained from 161 HIV-infected adolescents, 13-18 years of age, living in Constanta County, Romania. The cohort consisted of subjects diagnosed with HIV infection before 1995, who were followed up on a regular basis and who had no signs of acute or chronic hepatitis (absence of jaundice, fatigue, right upper quadrant pain, abdominal distension, nausea, poor appetite and malaise; and absence of abnormal biochemical data: alanine and aspartate aminotransferase less than twice the upper limit of normal, normal serum levels of gamma glutamyltransferase, alkaline phosphatase, albumin, and bilirubin). One hundred twenty-six patients (78.2%) were in US Centers for Disease Control and Prevention (CDC) clinical category B and only 35 were in category C; 79.5% were receiving antiretroviral therapy. A blood sample was obtained from each subject every 6 months during 2002 and 2003. A control group was established, consisting of 356 age-matched, healthy, HIV-uninfected adolescents, living in Constanta and 2 neighboring counties, from whom a blood sample was obtained during the same period as part of a seroprevalence surveillance after a rubella catch-up vaccination program. Whole blood was collected by venipuncture into EDTA anticoagulant. Samples were centrifuged within 2 hours after collection, and plasma was transferred to cryovials, frozen at -80°C and stored at the Institute of Virology, Bucharest. Laboratory personnel were not aware of the clinical status of the adolescents from whom blood was obtained. The study was approved by review boards for human subject research in Romania and at Baylor College of Medicine, Houston, Texas. Informed consent was obtained from each subject's parent or legal guardian.

Hepatitis markers were tested by immunoenzymatic assays (Murex Biotech Limited; Kent, England) and nucleic acid amplification tests (Roche Molecular System, Inc; Branchburg, New Jersey). All sera were initially tested for total antibody against hepatitis B core antigen (HBcAg), for antibody to hepatitis B surface antigen (HBsAg), and for antibody against HCV. Sera positive for anti-HBc antibody were tested for HBsAg; sera positive for anti-HBs antibody were retested in a quantitative assay to determine the anti HBs titer (>10 mIU/mL is considered protective). Sera positive for HBsAg were tested for hepatitis B e antigen (Murex HBeAg/anti HBe) as a marker for active HBV replication. Detection of hepatitis B viral DNA was done using a commercial kit for quantitative in vitro nucleic acid amplification ( AMPLICOR HBV Monitor test). The linear range for the AMPLICOR HBV Monitor test is between 1 x 103 and 4 x 107 copies/mL, with a lower limit of detection of 1000 HBV DNA copies/mL.

Virologic and immunologic HIV infection markers were determined, each by an independent investigator, only for samples collected from HIV-infected patients. CD4+ cell counts and viral loads were performed on blood samples anticoagulated in EDTA solution. CD4+ cell counts were made using the COULTER Manual CD4+ Count Kit . This is a light microscopy-based assay, which depends on the ability of monoclonal antibody-coated latex spheres to bind to the surface of cells expressing discrete antigen determinants. Plasma HIV-1 RNA quantification was performed with a commercial nucleic acid amplification test ( AMPLICOR HIV-1 MONITOR TEST version 1.5 ). Quantitative measurement of plasma HIV RNA levels was expressed in the number of HIV RNA copies/mL (lower detection limit, 400 copies/mL; linear range, 400-750,000 copies/mL).

Differences in HBV marker prevalence rates were evaluated by Fisher's exact test analysis of contingency tables, using GraphPad QuickCalcs ( https://www.graphpad.com/quickcalcs/index.cfm ). Two-tailed P values were reported. P < .05 was considered statistically significant.

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