HCV and TTV Co-infection in Turin, Italy

Abstract

To the Editor Population-based studies of patients suffering from chronic hepatitis have shown that 40–60% are associated with hepatitis C virus (HCV) infection. Long-standing hepatocyte damage and chronic inflammation can eventually lead to fibrosis, cirrhosis, and hepatocellular carcinoma [1]. The possibility of HCV co-infection with other viruses is well known. Transfusion-transmitted virus (TTV) is found worldwide and its role either in causing liver disease or in significantly increasing the severity of coexistent HCV hepatitis is doubtful. Literature data reported a prevalence of HCV/TTV co-infection between 1% and 24% [2]. In our area (Regione Piemonte, Northwest of Italy, population of 4.5 million people), this aspect has not been investigated in depth. In a prospective study, conducted in the outpatient clinic [3] and laboratory of the Gastro-Hepatology Department of Molinette Hospital, Turin, which represents the main facility of Regione Piemonte dedicated to the management of liver diseases, we analyzed the prevalence of HCV/TTV co-infection. Sera of 91 consecutive patients (59 males; mean age 48.6 ± 11.3 years) with chronic HCV hepatitis, candidates to receive standard combination antiviral therapy (interferon and ribavirin), were examined at baseline. The diagnosis of chronic hepatitis was based on histology [4] and biochemical liver function tests [5]. HCV detection was performed using polymerase chain reaction (PCR) assays (Versant 3.0; Bayer Diagnostic Corporation, Tarrytown, NY, US; lower limit of detection 615 IU/ml. COBASAMPLICOR; Roche Diagnostic Systems, Branchburg, NY, US; lower limit of detection 50 IU/ml). TTV–DNA was extracted using a phenol-chloroform method as described by Casey et al. [6]. Briefly 50 ll serum was lysed by digestion overnight at 37 C with 1.1 mg proteinase K (Eurobio) in a final volume of 350 ll lysing buffer containing 60 mM ethylenediamine tetraacetic acid (EDTA), 60 mM Hepes pH 7.0, 200 mM NaCl, and 2.2% sodium dodecyl sulfate. A first phenol–chloroform extraction was followed by a second chloroform extraction, and nucleic acids were precipitated with cold isopropanol. DNA was pelleted by centrifugation and washed with cold 70% ethanol. The pellet was allowed to dry at room temperature and subsequently resuspended in 50 ll distilled water and stored at -20 C until further processing. To determine TTV–DNA a first round of amplification followed by a seminested PCR was performed with 10 ll extracted DNA according to Okamoto et al. [7]. Primers were designed to amplify well-conserved regions of TTV genome, allowing the detection of different genotypes and subtypes of TTV. The 271-bp amplification product of second-round PCR was analyzed by agarose gel electrophoresis and ethidium bromide staining. The v test and the Fisher exact test, when required, were used for statistical analysis, and a P value of less than 0.05 was considered significant. Positivity for TTV–DNA was found in 14 out of 91 (15.3%) patients. As reported in Table 1, no significant differences were found between co-infected versus not coR. Pellicano A. Olivero M. L. Abate A. Smedile M. Rizzetto Department of Gastro-Hepatology, Molinette Hospital, Turin, Italy URL: www.rinaldopellicano.blogspot.com