Journal of Medical Virology 83:989–995 (2011) Occult Hepatitis C Virus Infection in Iranian Patients With Cryptogenic Liver Disease Farah Bokharaei-Salim, 1 Hossein Keyvani, 1 * Seyed Hamid Reza Monavari, 1 Seyed Moayed Alavian, 2 Zahra Madjd, 3 Mohssen Nassiri Toosi, 4 and Amir Houshang Mohammad Alizadeh 5 1 Department of Virology, Tehran University of Medical Sciences, Tehran, Iran 2 Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Tehran, Iran 3 Department of Pathology and Oncopathology Research Center, Tehran University of Medical Sciences, Tehran, Iran 4 Department of Gastroenterology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran 5 Research Center for Gastroenterology and Liver Disease, Shaheed Beheshti University of Medical Sciences, Tehran, Iran The diagnosis of cryptogenic liver disease is made when after extensive evaluations, recog- nizable etiologies of chronic liver disease are excluded. In this study, the presence of hepatitis C virus (HCV) RNA was tested in peripheral blood mononuclear cells (PBMCs) taken from Iranian patients who although were found negative for plasma HCV RNA and anti-HCV antibodies, suf- fered from chronic liver disease of unknown eti- ology. From September 2007 to March 2010, 69 patients from Tehran with chronic liver disease of unknown etiology who were referred to our center were enrolled in the present study. PBMCs were isolated from 10 mL peripheral blood speci- mens. HCV-RNA status was tested in plasma and PBMCs samples by reverse-transcription poly- merase chain reaction (RT-PCR). HCV-RNA was detected in HCV-positive PBMCs specimens by RT-PCR method. HCV genotypes were sub- sequently analyzed in HCV-positive samples using restriction fragment length polymorphism (RFLP) assay; then HCV genotypes were con- firmed by sequencing of 5 0 non-coding fragments after cloning PCR products into pJET1.2/blunt cloning vector. HCV-RNA was detected in PBMCs specimens belonging to 7 (10%) out of 69 patients. Genotyping of the HCV-RNA isolated from PBMCs showed that 3 (43%) patients with occult HCV infection had genotype 1b, 2 (29%) had genotype 1a, and another 2 (29%) had gen- otype 3a. The results of this study suggest that patients with chronic liver disease of unknown etiology may have occult HCV infection in the absence of anti-HCV antibodies and plasma HCV-RNA. It has been suggested that in the absence of liver biopsy specimens, analysis of PBMC sample for HCV-RNA would be informa- tive. J. Med. Virol. 83:989–995, 2011. ß 2011 Wiley-Liss, Inc. KEY WORDS: occult hepatitis C virus infec- tion; HCV; peripheral blood mononuclear cells; chronic liver disease; Iran; cryptogenic INTRODUCTION In approximately 5% of patients with chronic liver disease, no etiology can be identified; such cases are known as cryptogenic liver disease. The diagnosis is made when after extensive evaluations recognizable etiologies of chronic liver disease such as viral hepatitis, autoimmune hepatitis, non-alcoholic steatohepatitis (NASH), metabolic liver disease, cytomegalovirus or Epstein-Barr virus infections, hepatotoxic drug, Wil- son’s disease, biliary tract disease, venous outflow obstruction, a 1 -antitrypsin deficiency, decompensated diabetes, thyroid dysfunction, alcohol abuse, iron over- load, morbid obesity, or other systemic diseases, are excluded. In reviewing of studies from Iran, hepatitis B virus (HBV) infection was found as the most common cause of cryptogenic liver disease [Honarkar et al., 2005; Hollinger et al., 2010]. Hepatitis C virus (HCV) is a single-stranded RNA virus of the family Flaviviridae and genus Hepacivirus. This virus infects over 170 million individuals world- wide and causes chronic hepatitis in up to 85% of cases. Grant sponsor: Tehran University of Medical Sciences; Grant number: 454. *Correspondence to: Hossein Keyvani, Department of Virology, Tehran University of Medical Sciences, Tehran, Iran. E-mail: keyvanlab@yahoo.com Accepted 14 December 2010 DOI 10.1002/jmv.22044 Published online in Wiley Online Library (wileyonlinelibrary.com). ß 2011 WILEY-LISS, INC.