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NDT Advance Access originally published online on February 19, 2007
Nephrology Dialysis Transplantation 2007 22(7):2027-2031; doi:10.1093/ndt/gfm028
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Hepatitis C, HCV genotypes and hepatic siderosis in patients with chronic renal failure on haemodialysis in Brazil

Kátia de Paula Farah1, Ricardo Andrade Carmo2, Carlos Maurício de Figueiredo Antunes3, José Carlos Serufo1, Vandack Alencar Nobre Júnior1, Lúcia Porto Fonseca de Castro4, Virgínia Hora Rios Leite4, Rogério Augusto Pinto Silva5, Maria Carolina Barbosa Álvares1, Guilherme Oliveira Corrêa6, Solange Cristina Uber Busek6 and José Roberto Lambertucci1

1Infectious Diseases Branch, Department of Internal Medicine, School of Medicine, Federal University of Minas Gerais, 2Minas Gerais State Blood Center (Fundação Hemominas), 3Department of Parasitology, Institute of Biological Sciences, 4Department of Pathology, School of Medicine, 5Division of Radiology, University Hospital, Federal University of Minas Gerais and 6René Rachou Research Center, Fiocruz, MG, Brazil

Correspondence and offprint requests to: Dr José Roberto Lambertucci, Infectious and Parasitic Diseases Branch, Internal Medicine Department, Medical School of the Federal University of Minas Gerais, Av. Alfredo Balena, 190, Santa Efigênia, Belo Horizonte, Minas Gerais 30130-100, Brazil. Email: lamber{at}uai.com.br



   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. The aim of this study was to investigate the HCV genotypes, hepatic siderosis, inflammatory activity and fibrosis of the liver in patients with chronic renal failure (CRF) on haemodialysis in Brazil.

Methods. A cohort of 72 CRF patients was compared with a group of 65 candidates for blood donation (CBD). For the subjects selected, who tested positive for anti-HCV antibodies and were HCV-PCR positive, a protocol with epidemiological, clinical and laboratory information was completed. An ultrasound-guided liver biopsy was performed and histological analysis of liver fragments was carried out. The presence of HCV-RNA in plasma was established by nested-RT-PCR. The genotype was determined by Restriction Fragment Length Polymorphism (RFLP) analysis of the PCR product.

Results. HCV genotype 1 was predominant in both groups, but genotype 2 was the second most common amongst CRF patients, and there was a significant difference when compared with the CBD group (P = 0.016). Regarding inflammation and fibrosis, no significant difference was observed in the histology of the liver between the study groups. Siderosis of the liver was more prevalent in the CRF group (P = 0.000). Severe complications of liver biopsies were reported in 10 CRF patients (13.2%).

Conclusions. Genotype 2 was observed more frequently in the haemodialysis group. No statistically significant difference was detected between the CRF and CBD groups with regard to both inflammatory response and liver fibrosis. Hepatic siderosis has been attributed to excessive iron administration. As percutaneous liver biopsy resulted in severe complications, we suggest that other procedures of evaluating liver damage in CRF patients should be looked at thoughtfully.

Keywords: chronic renal failure; genotyping; haemodialysis; hepatitis C; liver biopsy; siderosis



   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
The prevalence of hepatitis C virus (HCV) is higher amongst patients with chronic renal failure (CRF) on haemodialysis (HD) comparing with patients undergoing peritoneal dialysis and to the general population [1,2]. It is well known that the advent of screening of blood products for anti-HCV has virtually eliminated the transmission of HCV infection by blood product transfusions. Yet, the nosocomial transmission of the virus has been reported. Indeed, patients treated in HD units with a high prevalence of HCV infection are at increased risk of acquiring the infection.

The natural history of HCV infection in patients in HD units is peculiar owing, in no small part, to the difficulty of investigating a long-term disease in a population whose life span is shorter than that of the normal population [2–4]. Moreover, liver damage cannot be detected by straightforward laboratory analysis since levels of aminotransferases typically remain unaltered in most patients [2–6]. There are few studies correlating viral infection and liver histology in patients with end-stage renal disease [4,6]. The clinical significance of genomic diversity of HCV has yet to be completely elucidated. Several reports have emphasized genotype-related differences in disease severity, clinical outcomes and response to interferon in patients with chronic hepatitis C. However, the subject has not been explored sufficiently in patients who present CRF as comorbidity.

Herein we describe a transversal analytical study, performed between May 2000 and December 2003, involving 72 CRF patients and 65 candidates for blood donation (CBD), all of whom tested positive for HCV-PCR. The HCV genotypes, inflammatory activity and fibrosis of the liver, hepatic iron deposition and complications of liver biopsy were compared in the study groups.



   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Acknowledgements
 References
 
Subjects were selected from a cohort of patients with CRF who had been treated, for at least 6 months, in six HD units located in the metropolitan area of Belo Horizonte (MG, Brazil), and from an population anti-HCV positive of CBD population who were referred to the out-patient clinic of the Infectious Diseases Unit of the Federal University of Minas Gerais.

The study was approved by the Ethical Committee of the Federal University of Minas Gerais (Belo Horizonte, MG, Brazil). A written informed consent was obtained from each participant prior to commencement of the study.

Socio-demographic, epidemiological and clinical data
The background history recorded for each subject included the following: (i) age and gender; (ii) duration of HD; (iii) haemotransfusion history; (iv) use of inhalable or injectable illicit drugs; (v) record of schistosomiasis mansoni; (vi) previous occurrence of clinical acute hepatitis and/or jaundice; and (vii) previous HCV seroconversion. In addition, other risk factors related to hepatic disease were investigated including previous contact with hepatitis B virus (HBV), alcohol abuse (>40 g of alcohol/day) during the 6-month period preceding the study, the regular use of hepatotoxic drugs, a family history of hepatitis C, and clinical manifestations of hepatic dysfunction.

Criteria for selection of subjects
The inclusion criteria for subjects of both groups were (i) age between 18 and 65 years; (ii) two or more samples exhibiting a positive reaction to an anti-HCV enzyme immunoassay (EIA 3.0) over a period of >6 months; (iii) availability of at least one serum sample for the determination of alanine aminotransferase (ALT) during the period of study; (iv) negative reaction to HBV surface antigen (HBsAg); (v) negative reaction to anti-HIV-1/2 (EIA) and (vi) availability of liver biopsy samples suitable for histological examination and showing at least three portal tracts as revealed by haematoxylin eosin and trichrome stains of Masson and Gomori.

The general exclusion criteria were (i) previous or current treatment for hepatitis C; (ii) alcohol ingestion at levels > 20 g/day for females or 40 g/day for males detected during the study period; and (iii) clinical contra-indications for liver biopsy (i.e. class III or IV heart disease, severe malnutrition, ascites, prothrombin activity <75% of normal level, <70 000 platelets/ml, haemoglobin lower than 8 g/dl, a record of active or recurrent haemorrhage).

Serological analyses
Assay for anti-HCV antibodies
The presence of anti-HCV antibodies was determined using enzyme immunoassay EIA III (Abbott Laboratories, North Chicago, IL, USA) that detects antibodies against three different regions of HCV, namely, C100-3 (non-structural region 4), C33 (non-structural region 3) and C22 (putative core protein).

Genotyping and sequence analysis of HCV-RNA
For RNA extraction, blood samples were collected prior to heparin administration: serum samples were stored at –70°C. Serum RNA was extracted and HCV-RNA was detected by transcription followed by nested-RT-PCR using primers derived from the 5'-UTR non-coding region of the HCV genome as described by Chan et al. [7]. The genotype was determined by restriction fragment length polymorphism (RFLP) analysis of the PCR product as previously described [7]. In all experiments, appropriate control samples (i.e. no-cDNA, anti-HCV negative controls and RNase-treated positive controls) were analysed in parallel with test samples.

Histological analyses
Liver samples were obtained either by ultrasound-directed percutaneous biopsy, using a Tru-Cut® type needle (number 16G; diameter = 1.65 mm), or by video-laparoscopy. One liver sample was obtained from each of the study subjects for histological analysis. These samples were fixed with 10% formaldehyde solution and dehydrated with successively stronger solutions of ethanol, the solvent being finally removed with xylol.

Fixed tissues were embedded in paraffin, sectioned into 4–5 mm slices and, after removal of the paraffin, treated with haematoxylin eosin, Masson's and Gomori's trichrome stains (to differentiate between cytoplasmic and intercellular components) and Perls’ Prussian blue stain (to locate haemosiderin with Fe+++), and by silver impregnation to outline the reticular fibres.

A total of 53 patients (74.6%) on dialysis included in the CRF group and 50 individuals (79.4%) included in the CBD group presented more than five portal tracts in the liver biopsies. The development of steatosis in liver tissue was categorized as either absent, <33%, between 33% and 66% or >66%. The classification of hepatic siderosis followed that of Searle et al. [8], namely, absence of iron (0), iron granules visible at 400x magnification (I), iron granules visible at 100x magnification (II), iron granules visible at 25x magnification (III), and iron granules visible at 10x magnification or with the naked eye (IV). Histological data derived from liver biopsies were analysed using the METAVIR algorithm [9].

Statistical analyses
Preliminary descriptive analyses were performed to determine the frequency distributions of the categorical variables and the central tendencies of the continuous variables in both populations. Comparison between the CRF and CBD groups with regard to possible associations of selected variables was then performed. Chi-square and Fisher's exact tests were used to establish associations between the categorical variables. The Student t-test and the Kruskal–Wallis rank test were used to compare groups with respect to continuous normal and non-normal variables, respectively. Differences between groups were considered to be significant when P < 0.05.



   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Acknowledgements
 References
 
Socio-demographic, epidemiological and clinical data
The average age (±SD) of CRF patients was 43.6 ± 11.6 years, whilst that of CBD subjects was 39.6 ± 10.6 years (P = 0.04); subsequent analysis did not reveal age as a confounding variable. There was no significant difference between the CRF and CBD group for male/female preponderance. For the CRF group, the average time on HD was 7.0 (6.2–7.8 95% CI) years, and 31% (0.20–0.41 95% CI) of the patients presented a history of kidney transplantation with loss of implant function and subsequent return to HD. There was no evidence of clinical hepatitis in either group.

A history of haemotransfusion, of previous contact with HBV, of regular use of hepatotoxic drugs, and of family cases of hepatitis C was more frequent amongst the CRF patients than in the CBD subjects, although the incidence of other risk factors for HCV were more common amongst individuals of the latter group (Table 1). The data on total serum bilirubin, AST, serum albumin, gamma-glutamyltranspetidase, AFP and platelets is also depicted in Table 1.


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  		Table 1. Distribution of clinical and epidemiological characteristics and risk factors for HCV amongst 72 patients with chronic renal failure (CRF group) undergoing haemodialysis and 65 candidates for blood donation (CBD group), all HCV-PCR positive, during the period of May 2000 to December 2003

 
Genotyping data
In both groups, there was a predominance of genotype 1 (Table 2); however, genotype 2 was significantly more common in the CRF group (P = 0.016), and genotype 3 in the CBD group (P = 0.001).


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  		Table 2. Distribution of RNA-HCV genotype detected by PCR analysis of plasma samples amongst 72 patients with chronic renal failure (CRF group) undergoing haemodialysis and 65 candidates for blood donation (CBD group), all HCV-PCR positive, during the period of May 2000 to December 2003

 
Histological data
The histological characteristics of liver samples from CRF and CBD subjects are summarized in Table 3. Inflammatory activity was considered either absent (A0) or light (A1) in 87.3% of the CRF patients and in 81.5% of the CBD subjects (P = 0.226). Hepatic cirrhosis (F4) was detected in 2.8% of the CRF group and in 4.6% of the CBD group. Liver steatosis occurred in 15.5% of the CRF group and 39.7% of the CBD group (P = 0.004). Siderosis of Kupfer cells and hepatocytes was significantly more prevalent in CRF patients on HD (P = 0.000) than in CBD (Table 4).


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  		Table 3. Distribution of histological findings of liver samples, classified according to METAVIR, amongst 72 patients with chronic renal failure (CRF group) undergoing haemodialysis and 65 candidates for blood donation (CBD group), all HCV-PCR positive, during the period of May 2000 to December 2003

 

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  		Table 4. Distribution of location and degree of siderosis in liver samples amongst 72 patients with chronic renal failure (CRF group) undergoing haemodialysis and 65 candidates for blood donation (CBD group), all HCV-PCR positive, during the period of May 2000 to December 2003

 
Complications of liver biopsy
All CBD individuals were submitted to ultrasound-directed percutaneous liver biopsy and no complications resulted from the procedure. Most (84.5%) of the CRF patients on HD were submitted to ultrasound-guided liver biopsy, and in 15.5% the biopsy was performed during video-laparoscopy. No difference of complications was observed between the methods used (US or laparoscopy). Following biopsy, 10 CRF patients (13.2%) presented complications, some with more than one adverse event: one death occurred after massive intra abdominal bleeding; eight received transfusion of packed red cells; intravenous infusion of normal saline and glucose was given to all patients; cholecystitis in one case was treated with cholecystectomy; pancreatitis (one case); haemobilia (three cases) was treated with endoscopic sphincterotomy. Haemothorax was treated in two cases, with pleural drainage. All received opioid pain killers.



   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Acknowledgements
 References
 
Within the two groups, infection with HCV genotype 1 was the most prevalent. In the CBD and CRF groups, HCV genotypes 3 and 2 were the second most common, respectively. The difference in distribution of the HCV genotypes suggests the existence of distinct forms of disease acquisition and the possibility of nosocomial HCV transmission in the HD environment [10]. For example, five out of the eight CRF patients infected with HCV genotype 2 in the present study have been treated in the same HD unit. One patient in group 1 had steatosis (1.4%) and 12 in group 2 (18.5%). Steatosis was not correlated with genotype 3, nor with alcohol abuse (P = 0.5). Severe complications of liver biopsies were reported in 10 patients (13.2%) of the CRF group.

In the present study, no statistically significant differences were detected between the CRF and CBD groups with respect to both inflammatory response and liver fibrosis. This finding contrasts with the observations of others [11] who have proposed that liver disease may be less aggressive in individuals on HD. Hepatitis C morbidity can be affected by different risk factors, such us, co-infections, the duration of HCV infection and the immunological response of the host [12,13]. It is difficult to determine the time of infection in patients with hepatitis C and in our cases we have not evaluated the immunological status of the HCV infected patients in both groups.

A statistically significant difference was identified, however, between the CRF and CBD groups with respect to ALT (P = 0.0001) and AST (0.001) activities. It has been suggested that the normal reference levels for liver enzymes in the CRF population should be lowered in order to allow a better correlation with hepatic disease [2,5,14].

Age difference (marginally significant, P = 0.04) was observed between the study groups, but age was not identified as a confounder in further analysis; thus, age was not considered to be of clinical importance.

Currently, recombinant human erythropoietin (rHuEPO) is effectively used to treat anaemia in haemodialysis patients, and the administration of intravenous iron optimizes the response to rHuEPO thus increasing haematocrit levels. However, excessive use of this treatment may produce side effects owing to iron overload [15]. Herein, the prevalence of siderosis in Kupfer cells and hepatocytes was found to be significantly higher in the CRF group on HD compared with the CBD group. Since the treatment with haemotransfusion has been replaced in recent years by administration of rHuEPO, it is more likely to have been caused by the regular parenteral administration of iron. In our opinion, the doses of iron currently administered have been excessive, giving rise to an increased risk of hepatic lesions. We have not been able to demonstrate this association herein, but the subject merits further investigations.

We did not expect liver biopsy complications in the CRF group. The frequency of complications may in part be explained by platelet dysfunction already described in chronic haemodialysis [16,17]. Only two patients in the CRF group had complications after laparoscopy-guided liver biopsies and 8 out of 60 in ultrasound-guided biopsy (P = 0.92). Therefore, the procedure, used to obtain liver fragments, does not explain the large number of cases of bleeding in the CRF group described herein.

In short, the finding of HCV genotype 2 in the CRF population suggests that HCV infection in HD units can be attributed in part to nosocomial transmission, an aspect that clearly requires more attention. The complications resulting from liver biopsy indicate that alternative methods such as transjugular liver biopsy should be used in CRF patients. Serum markers of fibrosis should be considered in the future after its value in the diagnosis of fibrosis is confirmed [13,18,19]. Finally, based on our results, there is an urgent need to re-evaluate protocols for iron replacement in CRF and to investigate the importance of iron excess in the evolution of liver disease in patients on haemodialysis.



   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil and the Graduate Course on Tropical Medicine and Infectious Diseases, Universidade Federal de Minas Gerais, Brasil.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Memon MI, Memon MA. Hepatitis C: an epidemiological review. J Viral Hepatitis (2002) 9:84–100.[CrossRef][Web of Science][Medline]
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  3. Kalantar-Zadeh K, McAllister CJ, Miller LG. Clinical characteristics and mortality in hepatitis C-positive haemodialysis patients: a population based study. Nephrol Dial Transplant (2005) 20:1662–1669.[Abstract/Free Full Text]
  4. Hu KQ, Lee SM, Hu SX, et al. Clinical presentation of chronic hepatitis C in patients with end-stage renal disease and on hemodialysis versus those with normal renal function. Am J Gastroenterol (2005) 100:2010–2018.[CrossRef][Web of Science][Medline]
  5. Fabrizi F, Lunghi G, Finazzi S, et al. Decreased serum aminotransferase activity in patients with chronic renal failure: impact on the detection of viral hepatitis. Am J Kidney Dis (2001) 38:1009–1015.[Web of Science][Medline]
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  7. Chan SW, McOmish F, Holmes EC, et al. Analysis of a new hepatitis C virus type and its phylogenetic relationship to existing variants. J Gen Virol (1992) 73:1131–1141.[Abstract/Free Full Text]
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  10. Fabrizi F, Lunghi G, Guarnori I, et al. Hepatitis C virus genotypes in chronic dialysis patients. Nephrol Dial Transplant (1996) 11:679–683.[Abstract/Free Full Text]
  11. Meyers CM, Seeff LB, Stehman-Breen CO, Hoofnagle JH. Hepatitis C and renal disease: an update. Am J Kidney Dis (2003) 42:631–657.[CrossRef][Web of Science][Medline]
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  15. Fishbane S, Kalantar-Zadeh K, Nissenson AR. Serum ferritin in chronic kidney disease: reconsidering the upper limit for iron treatment. Semin Dial (2004) 17:336–341.[CrossRef][Web of Science][Medline]
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  17. Albuquerque W, Arantes V, de Paula Farah K, Lambertucci JR. Acute pancreatitis and acute cholecystitis caused by hemobilia after percutaneous ultrasound-guided liver biopsy. Endoscopy (2005) 37:1159–1160.[CrossRef][Web of Science][Medline]
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Received for publication: 17. 7.07
Accepted in revised form: 9. 1.07


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This Article
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