Objectives: Investigations into the viral causes of end-stage liver disease in liver transplant patients with cryptogenic underlying disease remain of interest. Hepatitis C virus infection, especially in its silent (occult) form, may play a key role in the introduction and development of cryptogenic cirrhosis. We aimed to determine the prevalence of occult hepatitis C virus infection in liver transplant recipients with cryptogenic cirrhosis.
Materials and Methods: In this cross-sectional study, 127 liver transplant recipients confirmed to have cryptogenic cirrhosis were included. Plasma samples of the patients underwent evaluation for hepatitis C virus antibody using the enzyme-linked immunosorbent assay method. Plasma samples and paraffin-embedded liver tissue samples were tested for hepatitis C virus RNA using nested reverse transcriptase-polymerase chain reaction.
Results: Hepatitis C virus RNA was detected in liver tissue sections of 10 patients (7.9%). However, none of the cryptogenic patients had hepatitis C virus RNA or antibody in their plasma samples. None of the patients had hepatitis C or G virus coinfection, but simultaneous detection of hepatitis B and hepatitis C virus was diagnosed in 4 liver tissue samples.
Conclusions: A finding of hepatitis C virus RNA in liver tissue samples of transplant recipients presents the historical possibility of occult hepatitis C virus infection as underlying disease in our patients with cryptogenic cirrhosis. Results present an important and determinative role of occult hepatitis C virus infection in the pathogenesis of cryptogenic cirrhosis, which needs further confirmation in additional studies.
Key words : End-stage liver disease, Hepatitis B virus, Occult infection
Viral infections are the primary cause of chronic liver diseases and orthotopic liver transplant (OLT) in many countries, including Iran.1,2 Hepatitis C virus (HCV), a blood-borne pathogen that infects 2% to 3% of the total world population,3,4 has been the primary cause of chronic liver disease, liver transplant, and hepatocellular carcinoma in the Western world.5 Iran has low endemicity for HCV infection,6 but lifestyle changes have increased the prevalence of this infection.7 For many years, hepatitis B virus (HBV) was the most common cause of chronic viral hepatitis in Iran, but the HBV vaccination programs combined with increasing seroprevalence of HCV infection could now result in HCV emerging as the primary cause of chronic viral liver disease over the next few decades.7 In addition, HCV genotypes 1 and 4 are the so-called difficult-to-treat genotypes.8 Unfortunately, most Iranian patients have been shown to carry these genotypes.7 Chronic HCV infection and liver cirrhosis could be diagnosed in up to 85% and 45% of infected patients, respectively.3,4
In Iranian patients with end-stage liver disease, the most common cause of OLT has been HBV followed by cryptogenic cirrhosis. Hepatitis C virus infection has placed fifth for liver transplant indication in Iran.1 Cryptogenic cirrhosis implies that no underlying cause of cirrhosis has been found by extensive clinical, serologic, and pathologic evaluations.9-11 Scientific advancements and devel-opment of new diagnostic tools have decreased the prevalence of cryptogenic cirrhosis. Although cryptogenic cirrhosis is not a frequent diagnosis in many countries (less than 5%), up to 30% of cirrhotic patients have been diagnosed with this indication.6,9,12
Occult HCV infection is described by the presence of HCV RNA in liver biopsy and/or peripheral blood mononuclear cells (PBMCs) of patients with plasma-undetectable HCV RNA in the absence or presence of anti-HCV antibodies.13-15 This type of detection is valuable in patients with cryptogenic liver cirrhosis.15 All HCV genotypes have the potential to create occult HCV infection.6,16
Liver transplant is still the only alternative for patients with end-stage liver disease, with cryptogenic cirrhosis as the fourth most common indication of OLT in the world. It has been estimated that about 10% of recipients receive liver transplants because of cryptogenic cirrhosis.11,17-19 Compared with other indications of OLT, patients with cryptogenic cirrhosis have poor outcomes and lower survival rates.20,21 Moreover, patients become reinfected with HCV,4 and HCV recurrence is the main cause of graft loss.22 Therefore, we evaluated the prevalence of HCV infection in Iranian cirrhotic patients who received OLT due to cryptogenic cirrhosis. Our aim was to determine whether there is a causal relationship between the occult form of HCV infection and diagnoses of cryptogenic cirrhosis.
Materials and Methods
In this cross-sectional study, we included 127 patients with established cryptogenic cirrhosis, which was determined by comprehensive evaluations of clinicopathologic findings. Patients provided written informed consent before study inclusion.
Plasma and paraffin-embedded liver tissue samples were tested for HCV RNA using nested reverse transcriptase-polymerase chain reaction (RT-PCR). For the preparation of tissue samples, 3-μm-thick sections were cut and deparaffinized 3 times with xylene solution. After several washes with absolute ethanol, samples were dried at 50ºC and weighed. Viral RNA was extracted from plasma and depa-raffinized liver tissue samples with the use of the RNX-Plus kit (CinnaGen Inc., Tehran, Iran), and cDNAs were synthesized with the use of random hexamer primers and Moloney murine leukemia virus reverse transcriptase (CinnaGen).
To determine the HCV sequence, a standard nested RT-PCR technique was performed in a total volume of 25 μL in the presence of specific primers (Table 1). The first round of PCR contained 2 μL of cDNA, 1× PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, 0.1 μM of each primer, and 2.5 U of Taq DNA polymerase (CinnaGen). The thermo-cycling condi-tions were 95°C for 5 minutes followed by 25 cycles of denaturation at 94°C for 50 seconds, annealing at 55°C for 40 seconds, and an extension step at 72°C for 50 seconds, with a final extension step at 72°C for 3 minutes.
The PCR mix in the second round of the nested RT-PCR was the same as the first round, except that 1 μL of PCR product from the first round was used as a template, MgCl2 was increased to 2 mM, and the oligonucleotide primers were substituted. The temperature cycling of the second round was started with 95°C for 5 minutes followed by 35 cycles of 94°C for 40 seconds, 64°C for 35 seconds, and 72°C for 40 seconds, with finalization with extension at 72°C for 3 minutes. Finally, the PCR products of 225 bp were separated and monitored using electrophoresis on 1% agarose gels.
Statistical analyses were performed to determine associations between occult HCV infection and risk factors using SPSS software (version 15, SPSS Inc., Chicago, IL USA). P < .05 was considered to be significant.
Our cohort of 127 patients had a mean age of 34.9 ± 15.9 years (range, 4-67 y) and included 80 males (63%). The male-to-female ratio was 1.7. All cryptogenic patients were seronegative for antibody against HCV and human immunodeficiency virus. Table 2 presents patient characteristics and data regarding acute rejection and patient death.
Prevalence of hepatitis C virus infection in plasma and tissue samples
Although none of the cryptogenic patients had HCV RNA in their plasma samples, HCV RNA was detected in liver tissue sections of 10 patients (7.9%). We also tested liver tissue samples of HCV-positive patients for hepatitis G virus (HGV) and HBV coinfections. The results of PCR assays showed that none of the patients had HCV/HGV coinfections, but simultaneous detection of HCV/HBV was diagnosed in 4 liver tissue samples.
Age (P = .565), sex (P = .497), and blood groups (P = .524) of patients had no effect on rate of HCV infection. In addition, graft rejection (P = .549) and patient death (P = .509) were similar between HCV-positive and HCV-negative patients. However, because liver transplant patients were from different geographical regions of Iran and were transfused several times before and after transplant, statistical correlations could not be made between geographical distribution and history of blood transfusions with regard to occult HCV infection.
Cryptogenic cirrhosis is an indication for liver transplant in Iran.1 Previous reports from Iran have shown that HCV infection is gradually replacing HBV as the most common chronic viral hepatitis infection.7 In Iranian populations, cryptogenic cirrhosis is widely distributed and may be due to a number of factors, including occult HCV infection,16 which is defined as the presence of HCV RNA in hepatocytes and absence of HCV RNA in the serum.13-15
Occult HCV infection has been previously evaluated in Iranian patients with cryptogenic liver cirrhosis. Bokharaei-Salim and associates16 tested 69 cryptogenic patients and found 7 (10%) patients had occult HCV infection. Keyvani and colleagues6 found occult HCV infection in 8.9% of Iranian liver transplant candidates with cryptogenic cirrhosis. Patients who had an abnormal level of alanine transaminase for more than 1 year and were simultaneously negative for viral hepatitis (HCV antibody, HCV RNA, and HBsAg) were also evaluated for occult HCV infection. Regardless of the method used to evaluate occult HCV infection, our study confirmed these previous reports, showing occult HCV infection in 7.9% of cryptogenic patients.
The diagnosis of occult HCV infection depends on liver biopsy as the criterion standard. This invasive procedure is limited for high-risk patients.16 Alternative techniques for laboratory diagnosis of occult HCV infection have been proposed. One diagnostic method is based on the evaluation of whole blood and PBMCs for detection of HCV RNA.6,15,16,23 Several previous studies6,16,23 have examined PBMCs in patients with cryptogenic liver cirrhosis. In our study, we focused on the use of liver biopsies. Almost all studies that examined PBMCs in patients with cryptogenic cirrhosis showed higher rates of occult HCV infection than shown with liver biopsies.6,16,23-26 These controversial results may be because of limitations of the diagnostic methods. Despite severe complications of occult HCV infection, such as necroinflammation, fibrosis, liver cirrhosis, and hepatocellular carcinoma,6,27,28 Pardo and associates showed that a lower number of liver cells were involved and a milder disease occurred with occult HCV infection.27
Castillo and associates26 diagnosed HCV RNA in 57 of 100 (57%) liver biopsy specimens, a rate higher than our results. In contrast to our results, some reports have shown that HCV RNA could not recoverable from liver tissue or PBMCs of patients be with cryptogenic cirrhosis.29,30
Hypothetically, HCV RNA should be more accurately detectable in liver biopsies and explants from patients with impaired immune function, such as liver transplant recipients, than in immuno-competent patients. Therefore, the need to detect occult HCV infection that is not uncovered by routine diagnostic methods may have an important bearing on development of new screening strategies based on liver biopsies from explants and the use of modern therapeutic interventions against HCV to avoid recurrence pretransplant.14,31
In conclusion, our study showed that evaluation of occult HCV infections in patients with cryptogenic liver cirrhosis is necessary and should be considered. Moreover, the simultaneous evaluation of PBMCs and liver biopsies should be performed in future projects.
DOI : 10.6002/ect.2017.0332
From the 1Shiraz Transplant Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran; 2Medical Biotechnology Research Center, Ashkezar Branch,
Islamic Azad University, Ashkezar, Yazd, Iran
Acknowledgements: The authors thank the Shiraz University of Medical Sciences and Ashkezar Islamic Azad University for their support. The authors have no conflicts of interest to declare.
Corresponding author: Mohammad Javad Kazemi, Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran
Phone: +98 9131596213
Table 1. Primer Sequences of Hepatitis C Virus Used for Nested Reverse Transcriptase-Polymerase Chain Reaction
Table 2. Demographic and Laboratory Parameters of Liver Transplant Patients With Cryptogenic Cirrhosis