Fabrizi F, Marai P, Di Filippo S et al. Hepatitis C virus infection in kidney graft .... Pozzato G, K.aneko S, Moretti L et al. Different genotypes of hepatitis C virus are ...
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46.2 + 10.8 years (range, 19-67) (Table 1). The median duration of post-transplantation follow-up was 80.7±45.8 months (range 16-214). Of these, 72 received kidneys from cadaveric and one from living-related donors. Prior to renal transplantation, 71 patients were on chronic haemodialysis (HD) treatment and two on continuous ambulatory peritoneal dialysis (CAPD). Hepatitis B markers, including HBsAg, anti-HBc and anti-HBs, were assayed by radioimmunoassay using commercially available kits (Abbott Laboratories, North Chicago, Illinois, USA). Liver disease was defined as an increase in serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to more than twice the upper limit of normal. Acute liver disease was defined as an elevation in the serum AST and ALT levels on two or more occasions at least 2 weeks apart but for less than 6 months. Chronic liver disease was defined as a persistent elevation of AST and ALT for more than 6 months. The history of blood transfusion requirement was retrospectively evaluated.
using a recombinant immunoblot assay (4-RIBA; Ortho Diagnostic Systems and Chiron) with arbitrary rating of the intensity of the bands from 1 + to 4 + . In this RIBA, antibodies against structural (c 22-3) and non-structural (c 33-c, c 100-3, and 5-1 -1) recombinant antigens were determined. The results of second-generation RIBA were evaluated, as suggested by other investigators [23]. The response to the individual antigens was scaled comparing the antigen band intensity to the internal control band included on each strip. A signal of at least 1 + in at least two antigen lanes is required for a specimen to be 'positive'. Reaction of at least 1 + in only one lane is interpreted as 'indeterminate' and no bands as 'nonreactive'.
Prevalence of HCV RNA by RT-PCR
The presence of anti-HCV antibody was determined by a second-generation ELISA including antigens from the structural and non-structural NS3 and NS4 regions of the HCV genome (IgG anti-c 22-3, anti-c 200). Semiquantitative confirmation of ELISA-positive samples was performed by
We used a simple and rapid method for reverse transcription (RT)-PCR amplification of HCV RNA from human serum that does not require the RNA extraction step. After denaturation (30 s at 92°C), 3 ul of serum were directly added to the RT mix (50 mM Tris HC1, pH 8.2, 70 mM KC1, 10 mM MgC12, 4 mM DTT, 12 units of human placental ribonuclease inhibitor (HP-RI), 0.4% Nonldet P-40, 50pmoles of a specific antisense primer (AJ), 250 mM dNTPs, 6 units of AMV-RT, in a final volume of 25 nl). RT was carried out at 42°C for 60 min, and the cDNA was added directly, after denaturation (5 min at 100°C), to the PCR mix. Amplification was performed following the nested primer protocol, as previously described [24], with two sets of primers located in the 5' untranslated (UTR) region of the viral genome [25]. The first round of PCR (35 cycles) was carried out using primers Al (5'-GATGCACGGTCT ACGAGACCTC-3) and SI (5'-AACTACTGTCTT CA CGCAGAA-3'), generating a PCR product of 289 bp. For the second round (25 cycles), we used primers A2 (5-GCGACCCAACACTACTCGGCT-3) and S2 (5'-ATGG CGTTAGTATGAGTG-3') generating a PCR product of 187 bp. PCR cycles were as follows: denaturation at 94°C for 1 min, annealing of primers at 45°C for 1 min, and elongation at 72°C for 2 min. PCR products were analysed by gel electrophoresis on 2% agarose gel and visualized under UV light after staining with ethidium bromide. Band sizes were assessed by direct comparison with standard molecular weight markers.
Table 1. Demographic, clinical, biochemical and serological data of the 73 kidney transplant recipients studied
HCV genotyping
Histological evaluation Eleven of 73 patients underwent liver biopsy. All patients were informed of the possible risks and benefits of the procedure. Samples of liver tissue were obtained by transparietal biopsy. Liver biopsy specimens were fixed in buffered formalin, processed to paraffin and routinely stained by the following methods: H&E diastase/periodic acid-Schiff, Perls' stain for iron. When present, hepatitis was classified as chronic after it had been present for 6 months, as determined clinically and histologically. The chronic lesions were further classified as chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), or chronic lobular hepatitis (CLH). Overall histological diagnoses were made using standard criteria [22]. An ti-HC V assays
Characteristics
Data
No. of patients
73
M/F
42/31 46.2±11
Mean age (years) Kidney transplant First Second Median biochemical parameters at the time of study ALT(U/1) AST(U/1) Patients with abnormal ALT and AST Serological parameters at the time of study HBsAg positivity HBsAb and/or HBcAb antibody positivity Clinical outcome Duration of follow-up (years)
70 3
21 (13-193) 23(11-293) 7 7 12
6.7±3.8
The HCV genotypes in the serum samples were determined using Amplicor HCV test (Roche Diagnostic System, Nutley, NJ) [26] and a hybridization assay, called line probe assay (LiPA, Innogenetics, Zwijndrecht, Belgium), based on the reverse-hybridization principle [27]. For the Amplicor HCV PCR kit we followed the manufacturer's instructions. Briefly, RNA was extracted from 100 ul of serum with the lysis buffer containing guanidine thiocyanate and 2-mercaptoethanol in the presence of RNA carrier; RNA was then recovered by isopropanol precipitation. HCV RNA was reverse transcribed and amplified in a single-tube reaction using a ready-to-use master mix containing Tth DNA polymerase, AmpErase, and primers KY 80 (sense 5' GCA GAA AGC GTC TAG CCA TGG CGT 3') and KY 78 (antisense 5' CTC GCA AGC ACC CTA TCA GGC AGT 3') containing a biotin residue at the 5' terminus to allow detection of amplification product. The conditions for RT-PCR were as follows: one cycle at 50°C for 2 min, 60°C for 30 min, 95°C for 1 min; two cycles at 95°C for 15 s, 60=C for 20 s;
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Virology and histology of HCV in kidney transplant recipients
38 cycles at 90°C for 15 s, 60°C for 20 s; one cycle at 60°C for 4 min; samples were soaked at 72°C until use. Amplification products, after alkaline denaturation, were hybridized to probe KY88 (5' GTT GGG TCG CGA AAG GCG TTG TGGT 3'), immobilized in wells of microtitre plates. After the addition of avidin-peroxidase conjugate and chromogen substrate, the absorbance was read at 450 nm. Samples showing optical density (OD) values exceeding 0.4 were considered positive. Negative and positive controls were included in each run. LiPA technology allowed the determination of the five major genotypes and six subtypes. Specific oligonucleotide probes immobilized as parallel lines on membrane strips were hybridized with amplified sample material (samples processed by Amplicor HCV PCR kit). During amplification, biotinylated primers were incorporated in the amplified DNA fragments. After hybridization, streptavidin labelled with alkaline phosphatase was added and became bound to any biotinylated hybrid previously formed. Incubation with NBT/BCIP chromogen resulted in a purple/brown precipitate. The reactivity of an amplified fragment with one or more lines on the strip allowed the recognition of the genotype of HCV.
Statistical analysis Chi-square test with a Yates correction factor and the Student's t test were used for statistical analysis of the univariate variables. Values were expressed as mean± standard deviation. A multiple logistic analysis (method: forward stepwise) was performed using SPSS program with anti-HCV positivity as the dependent variable; gender, age, age at transplant, duration of dialysis treatment before kidney transplantation, transfusional requirement, number of transplants received, positivity for HBV markers, graft survival, underlying nephropathies and aminotransferase levels were predictor variables. Statistical probability of less than 0.05 was considered significant.
Detection of HCV RNA by RT-PCR Eleven (73%) of 15 anti-HCV positive patients showed HCV RNA by RT-PCR in the serum. We detected no HCV RNA-positive seronegative patient. HCV genotyping INNO LiPA HCV analysis on the PCR products demonstrated that HCV subtypes 2a (« = 7), lb (n = 3), la (n = \) were present in our patient population. No patient was infected with different HCV subtypes. As for each antibody response toward the four HCV antigens, antibody to c 22-3 was found irrespective of the HCV genotype. It was found in all patients with HCV subtype 2a, lb, and la. In contrast, the prevalence of antibodies to NS4 proteins (5-1-1 and clOO) was very low in patients with HCV subtype 2a (2/7 = 28%), lb (1/3 = 33%) and la (0%). Most patients with genotype 2a (4/7 = 57%), lb (2/3 = 67%) showed antibody to c 33-c. There was no association between detectable HCV viraemia and seropositivity for HCVspecific antigens (Table 3). Biochemical signs of liver disease
There were 18 (25%) of 73 renal allograft recipients with signs of liver disease; 13 (72%) patients of 18 developed chronic liver disease; five (28%) showed signs of acute liver disease (Table 4). Table 3. HCV genotypes and 4-RIBA reactivities in ELISA-2 positive patients Patient no.
HCV subtype
ELISA reactivity
RIBA 2 5-1-1
Results Prevalence and risk-factors for anti-HCV antibodies There were 16 anti-HCV positive and 57 anti-HCV negative patients by ELISA screening test; 15 (94%) of them were reactive by 4-RIBA (10 females, 5 males), one was 4-RIBA negative. The confirmation by 4-RIBA of 16 ELISA 2 positive assay is shown in Table 2. The prevalence of anti-HCV positivity was 20% (15/73). Application of a logistic regression model to this group showed that gender (P=0.03) and aminotransferase levels were independent predictors of antiHCV positivity (P = 0.0004).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
2a 2a lb la 2a 2a lb lb 2a 2a 2a — — — —
clOO-3
c33
c22
Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos
Table 4. Assessment of biochemical signs of liver disease Table 2. Four-antigen recombinant immunoblot assay (4-RIBA) in 16 kidney graft recipients reactive by second-generation assay 4-RIBA 4-RIBA 4-RIBA 4-RIBA 4-RIBA
positive positive positive indeterminate negative
(reacting with four
RIBA antigens)
(reacting with three (reacting with two
RIBA antigens) RIBA antigens)
(reacting with one (not reacting with
RJBA anlifen) RIBA antigens)
Abnormal aminotransferase
4(25%) 0
Acute
Normal aminotransferase
Total
6 52
15 58
Chronic
5(32%) 6(37%) 1(6%)
HCV pos HCV neg
I 4
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We observed 17 (23%) of 73 patients with previous signs of liver disease; seven (9%) of 73 had raised aminotransferase concentrations at the time of this study. Among the 15 patients who were anti-HCV positive, nine (60%) showed raised hepatic enzyme levels; eight (89%) of nine showed chronic liver disease. Nine of 58 anti-HCV negative patients (15%) had elevated AST and ALT levels; five (55%) of nine developed chronic liver disease; the other patients developed acute liver disease. Median levels of raised serum AST and ALT concentrations in anti-HCV positive patients were 121.5 (80-662) and 127 (42-616) U/l respectively. Median levels of raised serum AST and ALT concentrations in anti-HCV negative patients were 108.5 (80-710) and 140 (80-120) U/l respectively. Six of 11 HCV RNA positive patients had raised enzyme hepatic levels, 12 of 62 HCV RNA negative patients had raised aminotransferase levels; the difference was significant (54% vs 19%, P = 0.QO3). Five of 73 kidney graft recipients showed biochemical signs of liver damage without signs of HBV and HCV infection. Three of 73 patients were positive for both antiHCV and HBsAg; one patient was positive for both anti-HCV and anti-HBc antibodies. These patients showed normal aminotransferase levels and did not undergo liver biopsy. Histological features of liver disease and HCV genotyping Eleven of 73 patients underwent liver biopsy. Three anti-HCV negative patients experiencing elevations in aminotransferase levels underwent liver biopsy: one patient demonstrated only mild steatosis, two patients showed findings of active and persistent chronic hepatitis. Eight anti-HCV positive patients underwent liver biopsy. There was one patient infected with HCV subtype la who demonstrated biochemical signs of chronic liver disease with histological features of lobular chronic hepatitis. Three patients were infected with HCV subtype lb: one patient did not show raised aminotransferase levels and did not undergo liver biopsy, two patients had raised aminotransferase concentrations with histological features of lobular chronic hepatitis with mild necroinflammatory activity and cholestasis of variable degrees. There were seven patients infected with HCV subtype 2a; four patients did not show raised aminotransferase concentrations levels and did not undergo liver biopsy; three patients had raised hepatic enzyme levels, with histological findings of persistent chronic hepatitis in two cases and chronic lobular hepatitis in the other case. Two patients were anti-HCV positive with raised AST and ALT levels, but they had no detectable HCV RNA in the serum; they showed histological features of active chronic hepatitis with very mild activity. No patient demonstrated severe CAH or developing cirrhosis or
F. Fabrizi el al.
established cirrhosis. No patient had severe disturbance of liver architecture. No hepatocellular carcinomas (HCCs) were seen in any of the biopsy specimens. Discussion Using a second-generation anti-HCV assay, 20% of our patients were anti-HCV positive. This is consistent with reports from other centres in which 6% [6] to 57% [7] of renal transplant recipients showed antiHCV antibodies. The use of a logistic regression model showed that gender and raised aminotransferase concentrations are independent predictors of anti-HCV positivity. Also in haemodialysis patients [28] and chronic renal failure patients on conservative therapy [29] attending our unit, HCV seemed to play a role in the development of liver disease. Interestingly the female patients have a higher frequency of HCV infection than males, which is in contrast to other reports. Indeed Alter et al. [30,31] observed that males had a higher rate of HCV infection than females; on the other hand it is as yet unclear whether there are gender-related differences in the natural history of this infection. Despite the availability of a wide variety of RT-PCR assays for HCV RNA detection, the reliability of RT-PCR for diagnosis of HCV viraemia remains a problem in numerous laboratories, as previously suggested by a European expert group on viral hepatitis [32]. We used a simple and rapid RT-PCR protocol for the determination of HCV RNA that enables a bypass of extracting viral RNA before amplification, a time-consuming step. This procedure proved to be sensitive, as previously reported [25]. We found a high prevalence (73%) of persisting viraemia in the anti-HCV positive patients of this highrisk group. Four (27%) of 15 transplant recipients were anti-HCV positive but did not show detectable HCV RNA. Numerous possibilities could account for this finding: the patients could have recovered from HCV infection or could have had fluctuating levels of viraemia or very low levels below the limits of detection of the RT-PCR protocol performed. Furthermore, the choice of oligonucleotide primers located in highly conserved genomic regions, such as the 5'-UTR, is critical for a clinically useful RT-PCR procedure. There is, indeed, a extreme variability of the viral genome, especially in the regions encoding the envelope viral proteins. Alternatively, a cause of underestimating the number of viraemic patients may be the possibility that HCV is only located in the liver or in other nonblood compartments; searching HCV RNA by RT-PCR analysis in liver biopsy samples should permit detection in some of these patients. The high prevalence of HCV RNA among antiHCV positive patients of our patient group (73%) suggests that antibodies against HCV are likely to reflect persisting viraemia in this high-risk population. Such a conclusion might not apply to a different high-
Virology and histology of HCV in kidney transplant recipients
risk group such as HD patients. We observed [33], in fact, a lower frequency of detectable HCV RNA in anti-HCV positive patients in chronic HD treatment at our unit—44%. The amount of immunosuppression that renal transplant recipients were receiving could permit an active viral replication and a high prevalence of HCV viraemia in these patients. It is noteworthy that raised aminotransferase concentrations were observed in 54% of the patients who had HCV viraemia and in 19% of the patients without detectable HCV RNA in serum (P = 0.003). Such finding shows the sensitivity of aminotransferase in detecting active viral replication in this high-risk group. On the contrary, we observed no association between detectable HCV viraemia and aminotransferase levels in our haemodialysis population [33]. Other investigators have also found that ALT values are a poor predictive test for HCV viraemia in HD patients [34] and kidney graft recipients [14-16]. On the other hand, 46% of the patients with HCV RNA in serum maintained normal biochemical liver function. In these cases a liver biopsy could morphologically characterize the presence of disease. In these patients, immunosuppressive therapy might, to a certain extent, suppress the degree of histological injury and make recognition of liver disease difficult on the basis of standard biochemical parameters such as AST and ALT levels. Other authors [35] previously showed histological evidence of chronic hepatitis in healthy blood donors with anti-HCV antibodies and normal aminotransferase concentrations. Five patients showed raised aminotransferase levels without signs of HAV, HCV, and HBV infection, which suggests that other aetiological agents (non-A, non-B, non-C) may be responsible for a proportion of cases of liver dysfunction in this patient population. We did not observe anti-HCV negative patients with positive RT-PCR results. This observation is in contrast with the findings of Lau et al. [13], who found in their study that 46% of HCV-infected recipients had no apparent antibody response to currently available antigens. A different duration of post-transplantation follow-up might justify this discrepancy. So far HCV genotyping could be achieved by large sequencing efforts in the coding region or in the 5' UTR, or by polymerase chain reactions on HCV cDNA with type specific sets of core primers, or by restriction fragment length polymorphism (RFLP) analysis in the 5' untranslated region or in the NS5 region. To investigate the distribution of HCV genotypes in our patient group, we used LiPA technology that allows an easy and fast determination of HCV types and subtypes according to the classification of Simmonds et al. [17]. There are, indeed, different HCV nomenclatures [17,36,37]. The classification system proposed by Simmonds et al. [17], based on sequence alignments of a large number of HCV sequences derived from several parts of the genome, is at present the most widely accepted. The HCV genotyping in our study showed that HCV subtype 2a is prevalent; this finding is in contrast to preliminary and very recent
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reports [38,39] that revealed a high frequency of HCV subtypes lb and la in northern and central Italy. On the other hand, these studies were mostly of other high-risk patient groups such as haemophiliacs and intravenous drug abusers. Our data do not reveal that subtype lb seems to be associated with more severe liver damage than the other subtypes, which is in contrast with very recent observations [40]. However, the number of patients examined is too low to draw firm conclusions. In conclusion, the prevalence of anti-HCV antibodies and HCV RNA was 20 and 15% respectively in renal allograft recipients attending our unit; there is a good association between anti-HCV and HCV viraemia; serum aminotransferase levels were good predictors of HCV viraemia in this population; HCV subtype 2a was predominant in our study group; all anti-HCV positive patients who underwent liver biopsy showed mild or moderate histological features of chronic hepatitis. Follow-up of greater than 10 years, including serial liver biopsies, will be necessary to evaluate the impact of HCV-related chronic liver disease on patients and graft survival. Acknowledgements. The authors are indebted to Prof. C. Ponticelli and staff of the Nephrology and Dialysis Division, Prof. A. Vegeto and staff of the Transplantation Surgery Division, and Prof. G. Sirchia and staff of the Blood Transfusion Center, at the Ospedale Maggiore Policlinico di Milano (Istituto di Ricovero e Cura a Carattere Scientifico, IRCCS), Milano, Italy for their contribution in the clinical management of patients.
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