Journals | Policy | Permission

This journal has moved to cii.elmerpub.com Please go to the new site to make submissions

Clinical Infection & Immunity

Original Article

Volume 6, Number 3, September 2021, pages 75-81

Prevalence of Hepatitis C Viremia in Heart Transplant Recipients: A Single-Center Experience

Overt hepatitis C virus (HCV) infection, one of the most common causes of chronic hepatitis and liver-related complications worldwide, is associated with positive anti-HCV antibodies (anti-HCV-Ab), detectable serum HCV-RNA and altered liver function tests. In contrast, occult HCV infection was hypothesised in patients with subclinical chronic liver damage in whom HCV-RNA could be detected using high-sensitivity-nested-reverse transcriptase polymerase chain reaction (hs-n-rtPCR) [1]. With this method, HCV-RNA was detected in 57% of patients with positive HCV-Ab but negative HCV-RNA by standard techniques. Many of these patients also had some degree of liver function test abnormality, suggesting the existence of a well-defined and atypical form of HCV infection [2]. The demonstration of HCV-RNA in the liver and in peripheral blood mononuclear cells suggests ongoing viral replication in these patients and could therefore be associated with liver inflammation and fibrosis [1, 3].

HCV persistence in an occult form could be related to specific conditions, such as immunosuppression and antineoplastic chemotherapy. Among the most profound and persistent immune compromise, there is the immune suppression of heart transplant recipients (HTRs) [4, 5].

Heart transplant (HTX) encompasses a high overall risk of viral infection/reactivation, including cytomegalovirus, Epstein-Barr virus and hepatitis B virus [6-9]. HCV-negative recipients of hearts from HCV-infected donors become HCV-RNA positive in most cases and may develop a severe, progressive liver disease [10]. A different outcome can be observed when a previously HCV-positive recipient undergoes HTX. In short (3 - 4 years) follow-up studies [11, 12], clinical outcomes of HCV-positive recipients without major liver disease were largely favorable. In a larger study with longer follow-up, most HCV-positive recipients developed chronic liver disease, and many progressed to cirrhosis and end-stage liver disease [13]. It is important to underscore that post-transplant HCV infection may translate into a higher risk of acute cellular rejection [14], de novo diabetes mellitus [15], may play a role in the pathogenesis of post-transplant lymphoproliferative disorders [16], and entails a higher risk of hepatocellular carcinoma [17]. Therefore, diagnosis of overt or occult, subclinical HCV infection may have prognostic relevance in the HTX setting.

Accordingly, the aim of this study was to estimate the prevalence of HCV-RNA positivity, even at trace levels, in a large cohort of HTRs and the de novo incidence of HCV infection post-transplant. Our analysis represents an attempt to investigate the existence of occult HCV infection in HTRs, irrespective of the presence of anti-HCV-Ab or elevated liver function tests.

Patients included in this study were divided into two different groups. In the retrospective group, 134 HTRs undergone transplant between 1988 and 2006 (denoted as TX-RET). For this group of patients, we were able to study both a pre-transplant and a single post-transplant serum obtained between some months and a few years after transplant. For 55 of these 134 patients, a serum sample from the organ donor was also studied. In the prospective group, HTRs were evaluated in a prospective fashion from first presentation due to advanced heart disease. Forty-five of these patients had subsequently been transplanted between 2007 and 2010, and were denoted as TX-PRO. For these 45 patients, one pre-transplant sample and several post-transplant samples were tested. No donor sera were available for these patients.

As controls, we studied two groups: 1) a cohort of regular, apparently healthy blood donors (n = 126), all showing negative HCV serology in multiple occasions (negative controls); and 2) a group of patients with known chronic hepatitis C screened for possible antiviral therapy (positive controls). The study protocol and procedures were approved by our Institutional Review Board. This study was conducted in compliance with the ethical standards of our institution on human subjects as well as with the Helsinki Declaration.

For the TX-RET group, serum samples had been already collected and stored in sterile cryovials at -80 °C. At least one pre-transplant sample, obtained at the time of transplant listing, and one post-transplant sample, obtained at a variable time after transplantation, were studied. For the TX-PRO group, blood samples were collected before HTX at the time of listing and subsequently thereafter at 2, 4, 8, 12, 24 and 48 weeks post-transplant. Further samples were obtained at yearly intervals after the first post-transplant year.

From stored serum samples, a 200 µL aliquot was used to extract viral RNA by the QIAmp MinElute Virus Spin kit. RNA samples were then subjected to amplification through our non-commercially available, nested, reverse transcriptase polymerase chain reaction showing a high sensitivity (hs-n-rtPCR, with a lower detection limit of about 10 copies/mL, equal to about 2 IU/mL). This hs-n-rtPCR was performed using primers for the 5’non-coding region (NCR) (One Step RT-PCR, Qiagen). For the nested PCR, two sets of primers were designed to amplify the genetic segment of interest: sense-5'-GTCTAGCCATGGCGTTAG-3' (nt. 76-93), antisense-5'-ACGGTCTACGAGACCT-3' (nt. 321-337), for the first round and sense-5'-GTGTCGTGCAGCCTCCAG-3' (nt 100-117) and antisense-5'-GGGGCACTCGCAAGCACC-3' (nt 300-317) for the second round. All RNA extraction and reverse amplification steps were performed with sterile material and under a laminar flow hood with HEPA filter, to prevent any potential contamination of samples. During samples’ handling, all recommended procedures to avoid false positive results were implemented [18].

To perform viral genotyping, we applied restriction fragment length polymorphism (RFLP) analysis, using restriction enzymes Hinf I and Hae III, to all positive PCR samples (Supplementary Material 1, www.ciijournal.org). By this RFLP analysis, we were able to discriminate among genotypes 1, 2 and 3 that represent about 99% of the overall genotypes circulating in our geographic area.

In order to confirm that the amplicons produced by nested PCR were belonging to HCV genome, we performed direct sequencing of 10 random PCR product samples using ABI Prism BigDye Terminator v3.1 ready reaction cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequence electrophoresis was performed in a 3730 DNA analyzer using a 50 cm capillary panel and a POP7 polymer (Applied Biosystems, Foster City, CA, USA). Sequence similarity of the amplicons was then evaluated by comparison with HCV sequences available in the Genbank database using an updated version of the BLAST software.

To corroborate the results of genetic assays, we also tested sera for the non-structural viral protein core (HCV core antigen), by means of an immunochemical assay on the Architect platform (Abbott Diagnostics, Germany), according to the manufacturer’s instructions. This assay shows a sensitivity of 3 fmol/L and detects core antigen irrespective of viral genotype and serologic status (i.e., presence or absence of HCV-Ab).

Finally, HCV-Ab were searched in study sera by means of an immunochemical assay on the Architect platform (Abbott Diagnostics, Germany), according to the manufacturer’s instructions.

The following parameters were assayed in our Hospital Central Laboratory after transplant: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), platelet count, white blood cell count and serum gamma-globulins.

Most analyses were descriptive and used median with total range to summarize numerical variables or number and percentage to describe qualitative variables. Differences between numerical variables in different subgroups were studied by one-way analysis of variance. Categorical data were compared with Fisher’s exact test. Statistical analyses were performed using the SPSS software v. 22 (Armonk, NY: IBM Corp.). A P value < 0.05 was used to denote statistical significance of the observed differences.

An illustration of the non-commercial hs-n-rtPCR used in this study is provided in Supplementary Material 2 (www.ciijournal.org). To confirm the specificity of our hs-n-rtPCR, we established whether the amplified band belonged to the HCV genome. This was accomplished by means of direct sequencing of the 262 bp band amplified in the I round of the RT-nested-PCR obtained in a random number of patients. Sequencing confirmed that the amplicon obtained in positive samples was clearly belonging to the 5’NCR of HCV genome, as per Genbank BLAST search (Supplementary Material 3, www.ciijournal.org). Indeed, except HCV, no other living organism sequence showed homology with the analyzed amplicon.

To evaluate sensitivity and specificity of our testing method, we assayed with hs-n-rtPCR 126 blood donors, of whom seven (5.5%) tested positive (P < 0.01 for comparison with TX-RET or TX-PRO). In contrast, all samples from 25 patients with known chronic hepatitis C tested positive for HCV-RNA using our hs-n-rtPCR.

Among TX-RET patients screened, 27/134 (20.2%) turned out to be positive for HCV-RNA in the pre-transplant serum. However, of these only 33.3% (9/27) also had a positive HCV-RNA result in a post-transplant sample, such that two-thirds (18/27, 66.7%) became negative during follow-up.

In contrast, of the 107 subjects who tested negative pre-transplant, 23/107 (21.5%) turned out PCR positive in a post-transplant sample. These results are summarized in Figure 1.

Click for large image

Figure 1. Results of qualitative HCV-RNA analysis of pre- and post-transplant sera in the TX-RET group. TX-RET: transplant-retrospective.

Qualitative HCV-RNA testing was performed also on sera from 55 organ donors. A substantial prevalence of positive HCV-RNA was found in this subgroup of mostly young subjects (4/55, 7.2%), with most (51/55, 92.8%) testing HCV-RNA negative. However, none of the four HTRs from HCV-RNA positive donors showed a positive sample during post-transplant follow-up.

Among TX-PRO patients screened, 10/45 (22.2%) turned out to be positive for HCV-RNA in the pre-transplant serum. Of these, only 30% (3/10) also had a positive HCV-RNA result in a post-transplant sample, whilst most (7/10, 70%) became negative during follow-up.

In contrast, of the 35 subjects who tested negative pre-transplant, nine (25.7%) turned out PCR positive in a post-transplant sample. These results are summarized in Figure 2.

Click for large image

Figure 2. Results of qualitative HCV-RNA analysis of pre- and post-transplant sera in the TX-PRO group. TX-PRO: transplant-prospective.

Among TX-RET patients, 40 of 50 (80%) HCV-RNA-positive samples showed presence of genotype 1 and 10/50 (20%) genotype 2. None had genotype 3 in this subgroup. Genotyping of the four heart donors with positive PCR showed an even distribution of genotypes 1 and 2.

Similar data were obtained in the TX-PRO group, where genotype 1 was found in 78.9% (15/19) and genotype 2 in 21.1% (4/19), with no genotype 3 positive cases.

HCV core antigen was tested on all available serum samples, and results are presented in Table 1. An absolute concordance was found between negative HCV-RNA on hs-n-rtPCR and absence of HCV core antigen. In contrast, antigen test was positive in only a proportion of HCV-RNA-positive samples. In particular, there were a numerically higher proportion of antigen-positive samples among HCV-RNA-positive samples after transplant (53%) compared to before transplant (33%) (P = 0.19).

Click to view

Table 1. Relationship Between HCV-RNA and HCV-Ag Positivity in the TX-RET Group and the TX-PRO Group

HCV serology was performed on all serum samples from TX-PRO patients, and was positive in one patient (2.2%) before transplant. This subject also tested positive for HCV-RNA. Of the remaining 44 patients with a negative pre-transplant HCV serology, nine (20.4%) were indeed HCV-RNA-positive. After transplant, four additional patients showed a positive HCV-Ab test at two different time points, and all of them also tested positive for HCV-RNA in post-transplant sera. There were however seven additional HCV-RNA-positive patients after transplant who remained negative for HCV-Ab. The concordance between HCV-RNA and HCV-Ab positivity in the TX-PRO group is shown in Table 2.

Click to view

Table 2. Relationship Between HCV-RNA and HCV-Ab Positivity in the TX-PRO Group, Before and After Transplant

We sought to determine the clinical implication of a positive HCV-RNA with hs-n-rtPCR, analyzing levels of ALT, AST and GGT measured after transplant in the TX-PRO group. As shown in Figure 3, patients with positive HCV-RNA showed overall a higher level of each of the three markers of liver damage. It is important to underline that none of these patients received any antiviral treatment during the study period.

Click for large image

Figure 3. Graphical representation of the levels of ALT, AST and GGT among TX-PRO patients. As shown from the box and whiskers plots, HTRs who tested positive for HCV-RNA also showed a significantly higher level of markers of liver damage. ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: gamma-glutamyl-transferase; TX-PRO: transplant-prospective; HTRs: heart transplant recipients.

Starting from the hypothesis that HCV can cause an occult infection [1-3], we assessed whether this occurred in HTRs. We choose this subset of patients because HTRs are heavily exposed to procedures at risk for blood borne infections, including HCV, and are kept under intense immune suppression [19]. We therefore aimed at proving the potential ability of the virus to persist, even in the absence of an antibody response, and subsequently cause occult liver disease in this clinical setting.

Analyzing the prevalence of positive HCV-RNA in HTRs, we observed a very high rate of infection. At variance with seroprevalence studies, suggesting an HCV infection rate of 7-18% [20-24], we show in two different and non-contemporary patient cohorts that HCV-RNA can be detected in sera from over 20% of HTRs. Interestingly, a similar figure was obtained when pre-transplant sera were assayed. However, not all those who tested positive for HCV-RNA before transplant were found to be positive when checked after transplant. Indeed, more than two-thirds of them showed negative HCV-RNA after HTX, despite of immune suppression initiation. Whether a cyclophilin B-mediated direct inhibitory effect of cyclosporin A [25, 26], universally taken by these patients, was in place, remains to be determined. On the other hand, a considerable number of subjects were found HCV-RNA-positive de novo after transplant (almost one in four). In this setting, immune suppression could have rather favored infection, but once again further study is needed. As previously mentioned, no effect of antivirals can be hypothesized as none of the studied HTRs received antiviral therapy.

Although these results appeared surprising to us, we had to acknowledge that amplified DNA was indeed belonging to HCV, as demonstrated by sequencing. Also, the reliability of our results was attested by the observation that the major biomarkers of liver damage were significantly higher in HCV-RNA-positive subjects compared to their negative counterparts. The consistency of results between the two patient groups studied also corroborates our findings. Finally, the HCV genotype distribution in both study subgroups was closely mirroring the local viral epidemiology of hepatitis C [27], with predominance of genotype 1 (about 80%) and genotype 2 (about 20%) and restriction of genotype 3 to people who inject illicit drugs [28, 29].

That the observed rate of HCV-RNA positivity was not due to chance is shown by the 5.5% prevalence found in our “negative control” group, i.e., healthy blood donors, a figure that indeed approximates that of the general population of our country [30]. This finding also resembled the one obtained in the HTX donor subgroup, mostly made of previously healthy and young subjects, further supporting the consistency of our results. It was interesting to observe that none of the HTRs from HCV-RNA-positive donors showed post-transplant HCV-RNA-positive samples, suggesting a low infectivity of patients with such a low level of viremia.

The relationship between HCV-RNA and HCV antigen (HCV-Ag) observed in this cohort deserves consideration. We found a very tight concordance between negative HCV-RNA and negative HCV-Ag, confirming that in the absence of viral genome, no transcription/transduction can occur. In contrast, only a fraction of HCV-RNA-positive subjects had detectable HCV-Ag, despite of the high sensitivity of the assay for the latter. This finding suggests the existence of a condition of very low level HCV viremia associated with no detectable viral protein and mostly negative serology - consistent with “occult” HCV infection. The fact that the prevalence of HCV-RNA positivity in our study was significantly higher in HTRs compared to blood donors suggests immune deficiency likely favors though is not a necessary event for this condition to occur.

The absence of HCV-Ab in most cases with a positive high-sensitivity HCV-RNA should also be discussed. In solid organ transplant recipients, absence of humoral immune responses is essentially the rule [31] and that is why we designed this study based on viral genome rather than viral antibody search. Also, we were mostly interested in the rate of de novo HCV infection after HTX, that may well occur in the absence of a detectable antibody seroconversion.

The limitations of our study include the relatively low number of patients/samples analyzed, and the lack of confirmation of results with a real-time, commercially available and approved HCV-RNA PCR assay. These weaknesses translate into a consequent lack of generalizability of our results and imply the need for further studies to better analyze the issue of occult HCV infection.

In conclusion, our results suggest at present that occult HCV infection may occur in HTRs irrespective of the immune status, although an immune compromise seems to amplify this phenomenon.

Suppl 1. Details of the Restriction Fragment Length Polymorphism Analysis, Using Restriction Enzymes Hinf I and Hae III.

Suppl 2. Example of 5’NCR RT-nested-PCR products run on an agarose gel electrophoresis. UV light revelation after 2% agarose gel electrophoresis of RT-n-PCR products. Lanes 1-2 = reaction controls; lane 3 = negative control; lane 4 = molecular weight standard marker, 50 bp; lane 5 = positive control; lanes 6-8 heart transplant patient samples. From lane 5 to 8 wells were loaded with aliquots of first round (261 bp) and second round (218 bp) PCR products.

Suppl 3. Panel A: Forward sequencing of the 262 bp amplicon. Panel B: Reverse sequencing of the 262 bp amplicon. Panel C: Alignment of 262 bp sequencing product. Sequence parts shown in yellow denote forward and reverse primers used. Nucleotides in red represent variations with respect to the original HCV sequence. Inconsistencies observed can be due to the high genetic heterogeneity of HCV.

Acknowledgments

Authors would like to thank for their collaboration Head Nurses Aniello Arpaia and Giovanni Fusco.

Financial Disclosure

This work was supported with internal research funds by the Second University of Naples, now University of Campania “L. Vanvitelli” and by the AORN Ospedali dei Colli.

Conflict of Interest

Authors have no conflict of interest to disclose relevant to the contents of this article. EDM received funding, personal fees or advisory board membership honoraria from Roche, Genentech, Pfizer, MSD, Angelini, Advanz pharma, Nordic pharma, BioMerieux, Medtronic, Trx, outside of this work.

Informed Consent

Patients gave their informed consent to data collection and anonymous storage.

Author Contributions

DI, RM, EF, and MG performed laboratory tests. IM and RZ obtained clinical data. FD’A built the electronic database. EDM designed the study and oversaw it. RZ and EDM analysed data. DI and EDM drafted the article. All authors read, commented and approved the final version of the article.

Data Availability

All data are available upon request from Dr. Domenico Iossa and Prof. Emanuele Durante Mangoni.

Abbreviations

HCV: hepatitis C virus; HCV-Ab: HCV antibodies; HTRs: heart transplant recipients; HTX: heart transplant; hs-n-rtPCR: high-sensitivity-nested-reverse transcriptase polymerase chain reaction; TX-RET: transplant-retrospective; TX-PRO: transplant-prospective; ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: gamma-glutamyl-transferase

1. Castillo I, Pardo M, Bartolome J, Ortiz-Movilla N, Rodriguez-Inigo E, de Lucas S, Salas C, et al. Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown. J Infect Dis. 2004;189(1):7-14.
   doi pubmed
2. Bartolome J, Lopez-Alcorocho JM, Castillo I, Rodriguez-Inigo E, Quiroga JA, Palacios R, Carreno V. Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C. J Virol. 2007;81(14):7710-7715.
   doi pubmed
3. Castillo I, Rodriguez-Inigo E, Bartolome J, de Lucas S, Ortiz-Movilla N, Lopez-Alcorocho JM, Pardo M, et al. Hepatitis C virus replicates in peripheral blood mononuclear cells of patients with occult hepatitis C virus infection. Gut. 2005;54(5):682-685.
   doi pubmed
4. Castella M, Tenderich G, Koerner MM, Arusoglu L, El-Banayosy A, Schulz U, Schulze B, et al. Outcome of heart transplantation in patients previously infected with hepatitis C virus. J Heart Lung Transplant. 2001;20(5):595-598.
   doi
5. Lunel F, Cadranel JF, Rosenheim M, Dorent R, Di-Martino V, Payan C, Fretz C, et al. Hepatitis virus infections in heart transplant recipients: epidemiology, natural history, characteristics, and impact on survival. Gastroenterology. 2000;119(4):1064-1074.
   doi pubmed
6. Durante-Mangoni E, Andini R, Pinto D, Iossa D, Molaro R, Agrusta F, Casillo R, et al. Effect of the immunosuppressive regimen on the incidence of cytomegalovirus infection in 378 heart transplant recipients: A single centre, prospective cohort study. J Clin Virol. 2015;68:37-42.
   doi pubmed
7. Gasink LB, Blumberg EA, Localio AR, Desai SS, Israni AK, Lautenbach E. Hepatitis C virus seropositivity in organ donors and survival in heart transplant recipients. JAMA. 2006;296(15):1843-1850.
   doi pubmed
8. Pfau PR, Rho R, DeNofrio D, Loh E, Blumberg EA, Acker MA, Lucey MR. Hepatitis C transmission and infection by orthotopic heart transplantation. J Heart Lung Transplant. 2000;19(4):350-354.
   doi
9. Vitrone M, Iossa D, Rinaldi L, Pafundi PC, Molaro R, Parrella A, Andini R, et al. Hepatitis B virus reactivation after heart transplant: Incidence and clinical impact. J Clin Virol. 2017;96:54-59.
   doi pubmed
10. File E, Mehra M, Nair S, Dumas-Hicks D, Perrillo R. Allograft transmission of hepatitis C virus infection from infected donors in cardiac transplantation. Transplantation. 2003;76(7):1096-1100.
    doi pubmed
11. Cano O, Almenar L, Martinez-Dolz L, Moro J, Izquierdo MT, Aguero J, Sanchez R, et al. Course of patients with chronic hepatitis C virus infection undergoing heart transplantation. Transplant Proc. 2007;39(7):2353-2354.
    doi pubmed
12. Fujisaki T, Mikami T, Kuno T, Moss N, Itagaki S. Effect of hepatitis C virus infection on heart transplants in the current era. Transplantation. 2021.
    doi pubmed
13. Fagiuoli S, Minniti F, Pevere S, Farinati F, Burra P, Livi U, Naccarato R, et al. HBV and HCV infections in heart transplant recipients. J Heart Lung Transplant. 2001;20(7):718-724.
    doi
14. Gidea CG, Narula N, Reyentovich A, Fargnoli A, Smith D, Pavone J, Lewis T, et al. Increased early acute cellular rejection events in hepatitis C-positive heart transplantation. J Heart Lung Transplant. 2020;S1053-2498(20)31624-7.
15. Bodziak KA, Hricik DE. New-onset diabetes mellitus after solid organ transplantation. Transpl Int. 2009;22(5):519-530.
    doi pubmed
16. Burra P, Buda A, Livi U, Rigotti P, Zanus G, Calabrese F, Caforio A, et al. Occurrence of post-transplant lymphoproliferative disorders among over thousand adult recipients: any role for hepatitis C infection? Eur J Gastroenterol Hepatol. 2006;18(10):1065-1070.
    doi pubmed
17. Hoffmann CJ, Subramanian AK, Cameron AM, Engels EA. Incidence and risk factors for hepatocellular carcinoma after solid organ transplantation. Transplantation. 2008;86(6):784-790.
    doi pubmed
18. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989;339(6221):237-238.
    doi pubmed
19. Weinfurtner K, Reddy KR. Hepatitis C viraemic organs in solid organ transplantation. J Hepatol. 2021;74(3):716-733.
    doi pubmed
20. Pereira BJ, Milford EL, Kirkman RL, Levey AS. Transmission of hepatitis C virus by organ transplantation. N Engl J Med. 1991;325(7):454-460.
    doi pubmed
21. Garcia G, Terrault N, Wright TL. Hepatitis C virus infection in the immunocompromised patient. Semin Gastrointest Dis. 1995;6(1):35-45.
22. Lunel F, Cadranel JF, Perrin M, Grippon P, Desruenne M, Huraux JM, Cabrol C, et al. Anti-hepatitis C virus (HCV) antibodies in heart transplant recipients with posttransplantation chronic viral B and non-A, non-B hepatitis. Dig Dis Sci. 1991;36(1):124-125.
    doi pubmed
23. Ong JP, Barnes DS, Younossi ZM, Gramlich T, Yen-Lieberman B, Goormastic M, Sheffield C, et al. Outcome of de novo hepatitis C virus infection in heart transplant recipients. Hepatology. 1999;30(5):1293-1298.
    doi pubmed
24. Zein NN, McGregor CGA, Schwab K. Prevalence of hepatitis C infection among heart transplant recipients. Hepatology. 1994;4:141.
25. Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, Shimotohno K. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell. 2005;19(1):111-122.
    doi pubmed
26. Heck JA, Meng X, Frick DN. Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase. Biochem Pharmacol. 2009;77(7):1173-1180.
    doi pubmed
27. Pafundi PC, Parrella A, Iossa D, Molaro R, Battimelli C, Falco E, Sodano G, et al. Viability of pegIFNalpha-RBV for CHC in the direct acting antiviral era: a practical algorithm between efficacy and cost containment. J Chemother. 2017;29(2):94-101.
    doi pubmed
28. Zampino R, Pisaturo MA, Cirillo G, Marrone A, Macera M, Rinaldi L, Stanzione M, et al. Hepatocellular carcinoma in chronic HBV-HCV co-infection is correlated to fibrosis and disease duration. Ann Hepatol. 2015;14(1):75-82.
    doi
29. Zampino R, Ingrosso D, Durante-Mangoni E, Capasso R, Tripodi MF, Restivo L, Zappia V, et al. Microsomal triglyceride transfer protein (MTP) -493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 infected patients. J Viral Hepat. 2008;15(10):740-746.
    doi pubmed
30. Chiaramonte M, Stroffolini T, Caporaso N, Coppola R, Craxi A, Gaeta GB, Sagnelli E, et al. Hepatitis-C virus infection in Italy: a multicentric sero-epidemiological study (a report from the HCV study group of the Italian Association for the Study of the Liver). Ital J Gastroenterol. 1991;23(9):555-558.
31. Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med. 2007;357(25):2601-2614.
    doi pubmed

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Clinical Infection and Immunity is published by Elmer Press Inc.