A study on the serologic and molecular prevalence of hepatitis G virus ( HGV ) and hepatitis C virus ( HCV ) infections in patients with thalassemia in Larestan of Iran

Hepatitis G virus (HGV) and hepatitis C virus (HCV) infections may have role in introducing or complicating the clinical outcome in patients with thalassemia. In this study, the molecular and serological prevalence of HGV and HCV infections were evaluated in patients with thalassemia and compared with healthy controls. In a cross sectional study, the samples were collected from 86 patients with thalassemia and 100 healthy controls. The anti-HCV and HGV antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) methods. Also, the HGV and HCV viremia were analyzed in patients with thalassemia and controls by multiplex-nested-real time-polymerase chain reaction (RTPCR) protocol. Our results showed that the anti-E2-HGV and HCV antibodies were found in 16 of 86 and 18 of 86 of thalassemia patients and none of controls, respectively. HGV viremia was diagnosed in 13 of 86 of patients with thalassemia and in 1of 100 of controls. HCV-RNA was diagnosed in 12 of 86 and 6 of 100 of thalassemia and healthy controls, respectively. The diagnosis of significant higher prevalence of HGV and HCV in patients with thalassemia compared with controls in this region of Iran emphasized the importance of these lymphotropic viral hepatitis infections in pathogenesis and outcome of thalassemia patients.


INTRODUCTION
β-Thalassemia or thalassemia major is the most common hereditary blood disorder in the Mediterranean basin especially in Iran that has affected 25,000 individuals and has 4 to 5% prenatal carrier rate in total population (Mirmomen and Alavian, 2005;Rahimi et al., 2008;Akhavan-Niaki et al., 2011).Multi-transfusion in patients with thalassemia necessary for patient survival is at high risk for transfusion-associated infectious diseases such as hepatitis viruses.Prevalence of viral hepatitis infections in thalassemic patients is related to the prevalence of the infections in the donor population and screening test technology used for blood and blood products (Ocak et al., 2006;Al-Sheyyab et al., 2001).Preventing transmission of these blood-borne viruses' remains a major preoccupation for transfusion services worldwide (Ocak and Kaya, 2006).Hepatitis viruses and lymphotropic flaviviruses, including hepatitis C and G viruses (HCV and HGV) may have important role in pathophysiology of Thalassemia.
Therefore, based on earlier studies and due to the little data available on the prevalence of hepatitis viruses especially HGV infection in patients with thalassemia in Larestan, the molecular prevalence of HGV and HCV infection was evaluated.

MATERIALS AND METHODS
The ethylenediaminetetraacetic acid (EDTA) treated blood samples were collected from two studied groups, including 86 patients with thalassemia who multi-transfused in Larestan Blood Transfusion Center and 100 healthy control group who clinically and laboratory ruled out any hematological abnormalities between year 2008 to 2010.The molecular and serological prevalence of HGV and HCV infections were analyzed in patients with thalassemia and compared with healthy controls.Also, some possible risk factors of thalassemia pathogenesis, including age, gender, marriage, and history of smoking, transfusion, surgery, bone marrow transplantation and HIV infection were statistically analyzed for all studied patients with non-Hodgkin's lymphomas (NHL).

HGV and HCV serological analysis
Antibody against E2 glycoprotein of HGV (Anti-E2 Ab) was evaluated in plasma samples by third generation ELISA kit (DIAPRO, Italy and Dade Behring Marburg, Germany), according to the manufacturer's instructions.Anti-HCV antibody (HCVAb) was also evaluated in the studied plasma samples by third generation ELISA kit (DIAPRO, Italy), according to manufacturer's instruction.

HCV-RNA extraction and amplification
The HGV and HCV-RNA genomes were extracted from plasma samples by RNX plus extraction procedure as previously described (Kao et al., 1997).The quality of extraction technique was evaluated by spiking of HGV and HCV-RNA in negative plasma samples.

Yaghobi et al. 5867 HGV and HCV cDNA synthesis
The presentation of HGV and HCV-RNA genomes was analyzed by an in-house HGV/HCV-nested-real time-polymerase chain reaction (RT-PCR) protocol.First, cDNA was sensitized from HGV and HCV genomes by in-house protocol as follow: 3 μL of viral extracted RNA was incubated at 25°C for 1 h and at 72°C for 10 min after treating with random hexamer and Moloney murine leukemia virus reverse transcriptase (M-MuLV-RT).The 20 μL RT-master mix contained 0.2 mmol of dNTPs, 0.01 mg/ml of random hexamer, 7.5 U/ml of M-MuLV-RT, 1 U/ml of ribonuclease inhibitor, and 4 μL of 5 μL RTbuffers.

HGV and HCV nested-PCR protocol
The PCR master mix of simple PCR contained 2 μl of cDNA, 0.1 pmol/μL of primers, 0.2 mmol of dNTPs, 2.5 U of Taq DNA polymerase, 2.5 μl of 10X PCR buffer, and 1.5 mmol of MgCl2.The 2 μl of simple PCR product was used in nested PCR step with the same condition as simple PCR mix.The total volume per reaction in the two rounds of simple and nested PCR protocol was 25 μl.The thermocycling conditions for simple and nested PCR rounds were the same.Simple PCR was initiated with a first round at 95°C for 5 min, followed by a second round of 25 cycles at 94°C for 50 s, 55°C for 40 s, and 72°C for 50 s, and finalized with extension at 72°C for 3 min.The nested PCR protocol was initiated with a first round at 95°C for 5 min followed by a second round of 35 cycles at 94°C for 40 s, 64°C for 35 s, and 72°C for 40 s, and finalized with extension at 72°C for 3 min.

Statistical analysis
Significant differences of serological and molecular diagnostic markers of studied HGV and HCV between thalassemia patients and controls and also statistical correlations between viral hepatitis diagnostic indices and possible risk factors of thalassemia were analyzed by use of parametric and non parametric analyses with SPSS for Windows (version 15, Chicago, IL, USA).A level of p≤0.05 was accepted as statistically significant.

RESULTS
In total, 41 of 86 (47.7%) patients with thalassemia were male and 45 of 86 (52.3%) of patients were female.Also 39 of 100 (39%) of controls were male and rest of them 61of 100 (61%) were female with average age of 36 years old.

Single and multiple infections of HGV and HCV
At least one of molecular and immunological markers of HGV and HCV was found in 23 of 86 (26.7%), and 28 of 86 (32.5%) of patients with thalassemia, respectively.In addition, HGV and HCV infective markers were found in 1 of 100 (1%), and 6 of 100 (6%) controls, respectively.Coinfection of HGV and HCV was diagnosed by evaluation of different markers of these two viruses in 11 of 86 (12.8%) of patients with thalassemia.Co-infection of HGV with HCV was not diagnosed in any controls.
The times of receiving blood was found in thalassemia patients with significant higher odds of positive HCVAb (p=0.0001) and positive HGVAb (p=0.0001).However, the significant higher odds of the viremia of HGV (p=0.08) and HCV (p=0.337) was not found with times of blood transfusion in studied patients (Table 2).

DISCUSSION
Iran is located in the thalassemia belt and about 10% of the population suffers from this hereditary hematological abnormality (Haghpanah et al., 2010).Particularly in Larestan, which is located in southern of Iran, the prevalence of this disorder is very high as a result of the close relative marriage.Some of blood-borne viruses are common and have determinative role in outcome of patients with thalassemia.Viral hepatitis infections, including HGV and especially HCV and their related hepatic and extrahepatic clinical manifestations are at the higher risk of interfering in the pathophysiology of thalassemia (Mirmomen and Alavian, 2005;Boroujerdnia et al., 2009).Therefore, in this study, the molecular and serological prevalence of hepatitis viruses was evaluated patients with thalassemia, which is similar to Italian patients (11%) and Greek patients with thalassemia (19%) (Cacopardo et al., 1998;Amini et al., 2005).But in other study, the HGV viremia was found in 9 of 402 (2.24%) multi-transfused thalassemia cases.In this report, HGV genome was significantly found in patients with thalassemia and controls (15.11 vs. 1%), respectively (Table 1).Significant higher odds of HGV viremia was found among patients with thalassemia (p=0.0001,OR=0.057, 95% CI=0.007-0.443)(Table 1).Also, Anti-E2 antibody was significantly higher prevalence in thalassemia patients versus controls (16.60% vs. 0%) (Table 1).Significant higher odds of positive HGVAb (p=0.0001) was found among patients with thalassemia (Table 1).In addition, significant higher odds of positive HGVAb (p=0.045) and positive HGV-RT-PCR (p=0.0001) was observed with age.Significant higher odds of a positive HGVAb (p=0.011) and positive HGV-RT-PCR (p=0.0001) was found among males with thalassemia.The times of blood transfusion was found in thalassemia patients with significant higher odds of positive HGVAb (p=0.0001), and significant higher odds of the viremia of HCV (p=0.337) was not found with times of blood transfusion in studied patients (Table 2).On the other hand, for familial relationships between HGV and HCV has been suggested that HCV and HGV are transmitted together (Cacopardo et al., 1998).In previous investigations, high prevalence of HGV and HCV co-infection was prevalent in patients with thalassemia (25%) (Amini et al., 2005;Desai et al., 2004).In this study, the co-infection of HGV and HCV was diagnosed in 12.8% of patients with thalassemia, but this co-infection was not seen in any controls.Finally, diagnosis of significant higher serological and molecular prevalence of single and co-infection of HGV and HCV in patients with thalassemia in Larestan of Iran, emphasized more on the importance of these lymphotropic viral hepatitis infections in introducing and complicating the clinical outcome of thalassemia patients.

Table 1 .
The prevalence of HGV and HCV infective markers in patients with thalassemia and controls.