Prevalence of occult hepatitis C virus infection in beta-thalassemia major patients in Ahvaz, Iran

Occult hepatitis C virus infection (OCI) is defined by the presence of HCV RNA in peripheral blood mononuclear cells (PBMCs) and liver tissue cells despite the absence of HCV RNA in plasma. Currently, OCI is classified into two types: seropositive OCI (anti-HCV positive and serum HCV RNA negative) and seronegative OCI (anti-HCV and serum HCV RNA negative). Beta-thalassemia is described as a blood disorder that decreases the synthesis of hemoglobin. Repeated blood transfusion is the standard treatment for patients with beta-thalassemia major (BTM), and this increases the risk of exposure to infectious agents. The aim of this study was to investigate the prevalence of OCI among BTM patients. Plasma and PBMCs were collected from 90 BTM patients who were referred to Shafa Hospital in the city of Ahvaz and were screened for HCV antibody using a commercial ELISA kit as the first step. Next, nested RT-PCR was performed on extracts of plasma and PBMCs. HCV RNA from positive PBMCs was sequenced, the sequences were aligned, and a phylogenetic tree was constructed to determine their relationship to reference sequences retrieved from the GenBank database. Seventy-nine out of 90 patients (87.8%) were negative for HCV Ab (seronegative), while 11 patients (12.2%) were seropositive. HCV RNA was found in PBMCs of four patients (66.7%) who were negative for HCV Ab (seronegative) and two patients (33.3%) who were positive for HCV Ab (seropositive). HCV RNA was not detected in plasma samples from these six patients. Six out of 90 BTM patients (6.7%) had OCI. HCV genotyping revealed that all six patients were infected with HCV subtype 3a. We found a high frequency of OCI in BTM patients, which warrants more attention, considering the importance of this infection. Further studies are needed to determine the actual prevalence of OCI in BTM patients in Iran.


Introduction
Hepatitis C Virus (HCV) is an enveloped positive single-stranded RNA virus belonged to the Flaviviridae family and Hepacivirus genus (1). HCV has been classified into eight genotypes with 86 subtypes (2,3). HCV infection is transmitted through contact with infected blood, blood transfusion, drug injection, sexual intercourse, surgery, and the parenteral route (4,5). The course of infection can range from asymptomatic and acute to chronic liver diseases, such as fibrosis and hepatocellular carcinoma (HCC) (6). It has been estimated that the global prevalence of HCV infection in adults is 2.5% (7). Studies conducted on the general Iranian population found the estimated HCV infection prevalence to be 0.5% (8).
Beta-Thalassemia is defined as an inherited blood disorder affecting the synthesis of beta-globin chains in hemoglobin (9). The disease is characterized by several complications, such as insufficient erythropoiesis and chronic hemolytic anemia (10). Currently, regular blood transfusion is used as the first choice for the management of beta-thalassemia, and also patients receive ironchelation therapy, or bone marrow transplantation and supportive measures (11). Therefore, blood transfusion carries a considerable risk of acquiring blood transfusion-associated viral infections, such as hepatitis C virus, hepatitis B virus (HBV), and also human immunodeficiency virus (HIV) (12,13).
HCV infection is the common cause of post-transfusion hepatitis between Iranian patients with beta-thalassemia major (14). Iran has high levels of thalassemic carriers (15). It has been reported that the prevalence of HCV among Iranian beta-thalassemia major patients is 19% (16).
In Iran, HCV genotype 1 was the predominant genotype with a rate of 55% followed by genotype 3 at 37%. Among patients with HCV genotype 1, subtype 1a was the most predominant subtype with a rate of 79%, followed by subtype 1b at 19% (17).
HCV is mainly hepatotropic, but peripheral blood mononuclear cells (PBMCs) are the extrahepatic sites of viral replication (18). It has been shown in some patients; the HCV genotype in PBMC is different from that in the plasma (19)(20)(21)(22)(23). The genotype of HCV RNA in extrahepatic sites is a major prognostic factor for the HCV infection treatment process (18,23). PBMCs are a favorite site for replicating HCV after treatment, at least with interferon treatment (19,24). It is argued that the main cause of relapse or reinfection of HCV infection after liver transplantation is HCV replication in extrahepatic sites (24).
Occult HCV infection (OCI) is a new form of HCV infection that is characterized by the presence of HCV RNA in the liver tissue or PBMCs in the absence of HCV RNA in plasma, with or without HCV antibodies (Anti-HCV). Two clinical forms, based on the presence of anti-HCV Abs: seropositive with normal liver enzyme levels and seronegative with high liver enzyme levels.
Although the most reliable method for diagnosing OCI in all patients is detecting the HCV genome in the liver, the detection of HCV-RNA in PBMC is an alternative approach when a liver biopsy is not available. The current method for diagnosing HCV in Iran is detecting anti-HCV antibodies by ELISA and the detection of HCV RNA by Nucleic acid amplification tests (NAT) in plasma, which is unable to detect OCI (8,25,26).
Patients with OCI are potentially infectious, and the infection may be involved in the development of cryptogenic liver cirrhosis, fibrosis, and HCC. Repeated transfusion and a lack of molecular screening tests of blood donations for the presence of HCV RNA in PBMCs could increase the likelihood of OCI (27). This research aims to determine the prevalence of OCI and to detect risk factors for this infection among Iranian beta-thalassemia major patients. Hence, it is critical to screen patients with beta-thalassemia undergoing blood transfusion to detect OCI cases.

Ethical issues
The ethics committee approved the current research by the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran with registration number OG-9734 and Ethical code IR.AJUMS.REC.1397.745. Patients were informed of the present research and obtained a written consent form before their enrollment.

Study population
In this cross-sectional analysis performed between January 2018 and March 2019, a total of 90 Iranian patients with beta-thalassemia major referred to Shafa Thalassemia Clinic enrolled.
Inclusion criteria included: 1) patients living in Ahvaz city, 2) patients with the mental capacity to give written informed consent, and 3) patients with beta-thalassemia who regularly receive at least one unit of blood per month. The exclusion criteria included: 1) patients who disagreed to follow the study, 2) patients positive for HIV antibody, 3) Hemophilic patients, and patients with other types of hemolytic anemia, such as α thalassemia, sickle cell anemia, and spherocytosis. Patients' medical profiles were checked for demographic information, clinical history, and finding laboratory characteristics.

Collection and preparation of the specimens
Approximately 7mL of the peripheral blood sample was obtained from the patients and collected into a sterile EDTA-containing vacutainer tube. The plasma was separated by centrifugation at 2500 rpm for 5 min and preserved at -80˚C until testing. The PBMCs of samples were isolated by

Serological and biochemical tests
The Anti-HCV antibody in plasma samples was tested by a third-generation commercial ELISA

Detection of HCV RNA by RT-Nested PCR
The complementary DNA (cDNA) was prepared using a cDNA synthesis kit (Yekta Tajhiz, Iran).
In short, one μl of 50 μM random hexamer and 13.4 μl of diethyl pyrocarbonate (DEPC) water were mixed to make a mixture 0.5 μg of RNA template. The mixture was incubated at 72°C for 5 minutes and cooled quickly on the ice. Then, four μl of 5X reaction buffer was mixed with one μl of deoxynucleotide triphosphate (dNTPs) (10 mM), 0.5 μl of the RNase inhibitor (20 units) 1μl , and reverse transcriptase (200 units). The mixture was added to the previous mixture and incubated at 37°C for 60 minutes. Finally, the reaction was halted by heating at 70°C for 5 minutes.
The prepared cDNA was deposited at -80 ° C for further testing.
To detect HCV RNA, the 5′-untranslated region (5′-UTR) of the HCV genome was amplified by reverse transcriptase nested polymerase chain reaction (RT-nested PCR) using specific primers. RT-Nested PCR amplification for the first round was carried out with a total of 25 μl consisting of 12.5 μl PCR master mixture, one μl of each forward and reverse primer, two μl of cDNA, and 8.5 μl of distilled water. In the second round, one µl product of the first-round was used as a template, and 9.5 µl distilled water was added to reach the final 25 µl volume. The first and second rounds of RT-Nested PCR reaction mixture was subjected to thermocycler (Peqlab, Germany) with the following thermal program: initial denaturation at 94ºC for 5 min; and then 35 cycles of 94ºC/ 30ʺ, 62ºC/ 45ʺ, 72ºC/ 30ʺ, and a final extension at 72ºC for 10 min. The amplicon of 252 bp length was detected by electrophoresis on 2% agarose gel as second-round PCR products. Then RT-Nested PCR was performed on PBMC extraction of patients with negative results at the previous step. The second-round PCR products on 2% agarose gel, identified as positive OCI results. To confirm positive OCI patients, HCV RNA was amplified using RT-Nested PCR with two primers set from the core region (30).

RT-Nested PCR for the core region
The positive samples for the 5'-UTR region were again tested for the HCV core region of the HCV genome by RT-Nested PCR. The following specific primers Including, SC2: GGGAGGTCTCGTAGACCGTGCACCATG, AC2: GAGMGGKATRTACCCATGAGRTCGC, S7: AGACCGTGCACCATGAGCAC, and 584: CCCATGAGGTCGGCRAARC were used (31).
Two µl of the template with the same volume of PCR reaction mixture as described above was subjected to thermocycler for 30 cycles. The cycling conditions were achieved as follows: 94⁰C for 4 min; 30 cycles at 94⁰C for 1min, 45⁰C for 1 min, 72⁰C for 2 min; and final elongation at 72⁰C for 7 min. The expected PCR product for the outer set and the inner set was 500bp and 420 bp. PCR product was subjected to electrophoresis on a 2% agarose gel, stained with DNA safe stain, and observed under ultraviolet light.

HCV genotyping/sub-typing using Nucleotide Sequencing and phylogenetic analysis
The purified products from the second round of PCR amplification in both the forward and reverse directions were sequenced using an ABI 3730 XL DNA sequencer (BIONEER's Custom Services and Confidence Intervals were used for comparing differences between groups. The P-value lower than 0.05 was considered statistically significant.

Results
The existing cross-sectional research enrolled 90 patients who suffered from beta-thalassemia major. Serological experiments revealed that 79 of 90 subjects (87.8%) were negative for anti-HCV Ab, while 11 participants (12.2%) were positive. The RT-Nested PCR results for the HCV 5'-UTR revealed that 24 cases (26.7%) in their plasma were positive for HCV-RNA, and hence, excluded from the study owing to overt HCV infection. As Table-1 Table 1.
HCV genotyping was carried out via the HCV 5'-UTR sequencing for all the positive OCI patients.
Analysis of the results indicated that all six patients had HCV genotype 3a ( Phylogenetic analysis of these six sequences and corresponding sequence recovered from GenBank (NC-009824) demonstrated that all six OCI isolated from Ahvaz were subtype 3a ( Figure-2).    study from Iran showed there was no significant association between ALT, AST, and the presence of OCI among beta thalassemia major patients (33,27). Ayadi et al reported bet -thalassemia major patients with OCI had normal levels of ALT and AST, although one and two cases were at the upper limits of AST and ALT, respectively and triglyceride and LDL levels were higher in patients with OCI, compared with non-OCI patients (32).
As the RT-nested PCR from the present study shows, overall, six out of ninety (6.7%) betathalassemia patients undergoing blood transfusion had OCI, of which two (33.3%) and four (66.66%) cases were seropositive and seronegative, respectively, based on the anti-HCV Ab findings. On the other hand, the risk of OCI among seropositive persons is seven times that of seronegative ones (OR=7). This difference is not statistically significant however is borderline (p-value= 0.088) ( Table 1). Furthermore, sequence analysis revealed that all six patients had HCV genotype 3a (Table-2). The sequences obtained in this project had a 100% similarity to strain NCVI/PK1 from Pakistan (JN588558) (Figure 2).
These findings are in accord with results reported by other studies investigating the OCI status among the Iranian beta-thalassemia major patients, which indicated a prevalence rate of 3.3%-6.3% for OCI among the population (27,32,33). In this way, Bastani  in that study, three (6.3%) of the subjects were also positive for OCI by RT-nested PCR, withal genotypes 1a and 3a were reported as the most common subtypes during HCV genotyping (33).
Similarly, Ayadi and co-workers in another study from Iran showed that six (3.3%) of 181 betathalassemia cases had HCV RNA in their PBMC samples, and reported as OCI patients.
Furthermore, HCV genotypes 1b (three subjects), 1a (two subjects), and 3a (one subjects) were identified in that study (32). As HCV genotyping results from the mentioned studies and the present work demonstrate, the HCV genotypes found among OCI patients are in line with the most common HCV subtypes in Iran; 1a, followed by 3a and 1b (34 OCI should be more considered in this group. Indeed, it seems that the likelihood of OCI should be considered not only in thalassemic patients but also in other individuals who are at an increased risk of blood transfusion-associated viral infections.

Conclusion
Our study showed the prevalence rate of 6.7% OCI without evidence of the presence of HCV RNA in beta-thalassemia patients. Concerning serious complications and the clinical importance of OCI in beta-thalassemia patients, sensitive diagnostic methods for identifying HCV RNA in the PBMC should be implemented for all thalassemia patients when a liver biopsy is not available. Further studies with larger samples are needed to obtain a better estimate of OCI and to reveal the significance and repercussions of OCI in patients with beta-thalassemia major.   The phylogenetic tree based on the nucleotide sequences of HCV 5'-UTR region obtained from PBMC samples from participants who suffer from betathalassemia major with OCI. The tree was constructed using MEGA X based on maximum likelihood under the Tamura Nei-model. 1000 bootstrap replicates measured the tree's precision. In black circles the isolations obtained in this study are shown ().In the black square, the reference sequence is shown.