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Hepatitis C virus density heterogeneity and viral titre in acute and chronic infection: a comparison of immunodeficient and immunocompetent patients
Journal ofHepatology 1996; 25: 599-607 Printed in Denmark All rights reserved Munksgoard Copenhagen
Copyright Q European Association for the Study of the Liver 1996 of Hepstology ISSN 0168.8278
Journal
Hepatitis C virus density heterogeneity and viral titre in acute and chronic infection: a comparison of immunodeficient and immunocompetent patients Jonathan P. Watson1’2, Debra J. Bevitt1,2 , Gavin P Spickett3, Geoffrey L. Toms2 and Margaret F. Bassendine’ Departments of ‘Medicine, 2Wrology and 31mmunology. University of Newcastle upon Tyne, Newcastle upon Tyne, UK
Background: Heterogeneities
in the buoyant density of hepatitis C virus RNA have been reported in different groups of patients, and have been attributed to differential binding of viral particles to p-lipoproteins and IgG, and the presence of hepatitis C virus nucleocapsids in circulation. It may be that hepatitis C virus density heterogeneity correlates with the severity of liver disease, hepatitis C virus RNA titre, and the immunocompetence of the patient. Methods and Results: We have analysed five immunodeficient patients (one with hypogammaglobulinaemia and selective IgA deficiency, one with Xlinked agammaglobulinaemia, three with common variable immunodeficiency) who have been acutely infected with the same batch of intravenous immunoglobulin contaminated with hepatitis C virus (genotype la). The course of hepatitis C virus infection in these patients was compared to one immunocompetent patient who presented with acute hepatitis C virus and progressed to chronic disease, and seven immunocompetent patients with chronic hepatitis C. Serum samples were analysed by differential flotation ultracentrifugation in NaCl solution (density 1.063 g/ml). The high and low density fractions were tested for the presence of RNA by RT-PCR. Serum samples were also quantified for
F
acute hepatitis C virus infection, acute hepatitis with jaundice occurs in only 10% of cases (l), and fulminant hepatitis is very rare (1,2). OLLOWING
Received 19 October 1995; revised 22 Febmmy: accepted 23 February 1996
Correspondence: Dr. J. P. Watson, Department of Virology, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Tel.: 0191222 7775. Fax: 0191 222 7775.
hepatitis C virus RNA (Amplicor HCV Monitor kit, Roche Diagnostic Systems). Three quarters of the acutely infected patients analysed presented with low density hepatitis C virus. Low density hepatitis C virus was absent in most chronic infections but persisted in two patients with common variable immunodeficiency. High density hepatitis C virus was detected in the chronic phase in all acutely infected patients in whom the disease persisted, and was present in all samples from PCRpositive patients with chronic infection. Immunodeficient patients had significantly higher hepatitis C virus RNA titres on presentation than immunocompetent patients, but there was no correlation between titre and clinical course of infection. Conclusions: Heterogeneities in the buoyant density of hepatitis C virus RNA have been identified in the patient groups studied. Low density hepatitis C virus is detected more often in acute infection and high density hepatitis C virus is detected more often in chronic infection. Despite acute infection via the same route of infection with the same hepatitis C virus strain, the five immunodeficient patients studied all followed a different clinical course. Key words: Common variable immunodeficiency; Immunoglobulin; P-Lipoprotein; Quantitation.
Most patients follow a subclinical course, and the majority progress to chronic hepatitis (3). The variation in host response and the high rate of viral persistence is poorly understood, and a number of factors including virus genotype, infecting viral dose, selective defects in host cellular and humoral immune responses, and rapid viral sequence evolution may play a role. Hepatitis C virus RNA titre differs in different 599
J. P. Watson et al.
patient groups, and has been reported to be higher in severe disease (4). However, it is unclear whether viral RNA is associated with infectious virus. Hijikata et al. (5) were able to differentiate infectious and non-infectious PCR positive serum by infection of chimpanzees, and reported that hepatitis C virus RNA titre did not correlate with degree of serum infectivity, but hepatitis C virus RNA in infectious serum partitioned at a low density by differential flotation ultracentrifugation. Differences in hepatitis C virus RNA buoyant density between infected individuals have been reported: low density virus has been detected in acute disease and asymptomatic blood donors, and high density virus has been detected in chronic disease (45). The differences in buoyant density have been attributed to differential binding to IgG (high density fraction) and P-lipoprotein (low density fraction) (6,7). The high density fraction has also been attributed to the presence of uncoated nucleocapsids in circulation (4), and this is supported by electron microscopic visualisation of particles of different buoyant densities from hepatitis C virus positive plasma. Immunoelectron microscopy following sucrose density gradient centrifugation of human plasma with high hepatitis C virus RNA titre reveals spherical flavivirus-like particles 55-65 nm in diameter in the low density fractions (1.14-1.16 g/ml) which specifically react with polyclonal and monoclonal anti-HCV envelope antibodies, and smaller core-like particles 30-35 nm in diameter in the high density fractions (1.23-1.27 g/ml) (8). Also, detergent treatment of the low density fraction (1.115 g/ml) extracted from human hepatitis C virus positive serum separated on a potassium bromide density gradient releases icosahedron-shaped particles 33 nm in diameter, which react with anti-HCV core murine monoclonal antibody (9) Direct investigation of the host responses which may drive these changes following acute infection is difficult, as patients are infected with different strains of the virus in varying doses, and duration of infection is often difficult to determine. Recently, a group of patients with a variety of immune deficiencies were infected by a hepatitis C virus contaminated batch of intravenous immunoglobulin (lo), providing an opportunity to study the outcome of a uniform infectious hepatitis C virus challenge in patients with well characterised immune defects. The response of five immunodeficient patients in Newcastle upon Tyne to infection with this contaminated batch has been studied and compared with acutely and chronically infected immunocompetent individuals, by analysis of the clinical course of the disease in each
patient, measurement of hepatitis C virus density heterogeneity following acute infection, and hepatitis C virus RNA quantitation.
Materials and Methods Patients included in the study Five immunodeficient patients were acutely infected during February 1994 with a batch of intravenous immunoglobulin contaminated with hepatitis C virus: three with common variable immunodeficiency, one with X-linked agammaglobulinaemia, and one with hypogammaglobulinaemia and selective IgA deficiency. One patient with common variable immunodeficiency remained hepatitis C virus RNA negative, and the patient with hypogammaglobulinaemia and selective IgA deficiency died of an unrelated illness. Serum samples were collected from the three remaining patients on presentation at the liver clinic and sequentially thereafter (0385: common variable immunodeficiency, serum collected 10-56 weeks post-infection, C980: common variable immunodeficiency, serum collected 17-54 weeks post-infection, C935: X-linked hypogammaglobulinaemia, serum collected 12-61 weeks post-infection). Sequential serum samples were also collected 9-65 weeks postinfection from an immunocompetent patient with acute hepatitis C virus. He presented with acute hepatitis C virus infection following an episode of needle sharing during intravenous drug abuse, and progressed to chronic disease. For comparative study, single serum samples were taken from seven immunocompetent patients with chronic hepatitis C who had not been treated with a-interferon. All samples were stored at -70°C in 100 ~1 aliquots prior to analysis. The mean age of the acute immunodeficient patients was 38.8k8.3 years (2 females, 3 males), the age of the acute immunocompetent patient was 28 years (1 male), and the mean age of the chronic immunocompetent patients was 39.6k5.6 years (7 males). Clinical investigations All patients were treated in the liver clinic at the Freeman Hospital, Newcastle upon Tyne. Sequential liver function tests were performed, and 8/8 immunocompetent and l/3 immunodeficient patients proceeded to liver biopsy for clinical purposes. All patients had hepatitis C virus serology tested by ELISA-2 (Abbott Laboratories, North Chicago, IL, United Biomedical, Hauppage, NY) and RIBA(Ortho Diagnostic Systems). Genotype data was available on all patients (kindly provided by Dr P Simmonds, Edinburgh University).
HCV density heterogeneity and viral titre
Differential$otation ultracentrifigation Serum samples were fractionated by differential flotation ultracentrifugation according to the technique described by Have1 et al. (11). Briefly, 50 pl patient serum was mixed with 8 ml NaCVKBr solution with a density of 1.063 g/ml, and centrifuged at 139,500xg at 14OC for 22 hours. After centrifugation, 1 ml of the low density (top) fraction and 1 ml of the high density (bottom) fraction was collected. The fractions were analysed for the presence of hepatitis C virus RNA by RNA extraction from 100 pl of each fraction, followed by reverse transcription and nested PCR using oligonucleotide primers PTl-4 to amplify part of the hepatitis C virus 5’ non-coding region as previously described (12). Negative controls were used for all reactions, and the guidelines of Kwok & Higuchi (13) were followed to minimise the risk of contamination. Hepatitis C virus RNA quantitation Quantitation of hepatitis C virus RNA in patients’ serum was carried out using the Amplicor HCV Monitor Test (Roche Diagnostic Systems, UK), according to the manufacturer’s instructions. Results were interpreted by selecting the highest dilution of the amplicon that gave an optical density (OD) between 0.5 and 2.0 on the hepatitis C virus microwells (rows A-E) and on the quantitation standard (QS) microwells (rows F-H). The OD for the QS was required to be above 0.5 in at least one well for the determination to be valid. The OD and dilution factors were entered into the HCV monitor.xls Microsoft Excel worksheet file (Roche Diagnostic Systems,
UK, Microsoft Corporation, USA) to compute titre in hepatitis C virus RNA copies/ml.
the
Results Clinical course and investigations Table 1 shows the serological, biochemical, and histological data on the patients who were analysed by differential flotation ultracentrifugation and hepatitis C virus RNA quantitation. Two patients from the study are not included in this table: one patient with common variable immunodeficiency was hepatitis C virus RNA negative with persistently normal liver function tests following intravenous infusion with the contaminated immunoglobulin, and the patient with hypogammaglobulinaemia and selective IgA deficiency died of an unrelated cause (empyema and sepsis) shortly after intravenous infusion with the contaminated batch of immunoglobulin. At post mortem 20 weeks post-infection, no evidence of hepatitis was found in the liver. DifSerentialJZotation ultracentrifigation for analysis of buoyant density of hepatitis C virus particles in vivo Sequential serum samples from the four acutely infected patients (three immunodeficient and one immunocompetent) together with single serum samples from the seven chronically infected patients were analysed by differential flotation ultracentrifugation. Three of the four acutely infected patients presented with low density hepatitis C virus RNA (Table 2). The immunocompetent patient (C162) and the patient with X-linked agammaglobulinaemia
TABLE 1 Clinical and serological details of immunocompetent and immunodeficient hepatitis C virus infected patients analysed. For acutely infected patients the peak alanine transaminase (ah) level is given, followed by the chronic alanine transaminase level at the end of follow up (normal alanine transaminase ~45 iull). Key for liver biopsy data: ND: not done, CH: chronic hepatitis, CIRR: cirrhosis. l*: Genotype 1 but subtype unclassified in this patient Patient
Sex
Genotype
ELISA-2
RIBA-
Alt (m/l)
Liver biopsy
Immunodeficient, acute hepatitis C virus infection F C885 48 M c935 28 F C980 36
la la la
NEG NEG NEG
NEG NEG +(l)C33
2900 to 13 259 to 48 70 to 95
ND ND CIRR
Immunocompetent, Cl62
acute hepatitis C virus infection M 28
1*
POS
+(4)
979 to 98
CH
Immunocompetent, C356 c5 C8 c37 C89 Cl36 c35
chronic hepatitis C virus infection M 43 M 41 M 43 M 29 M 45 M 43 M 33
la la lb lb lb 2b 3
POS POS POS POS POS POS POS
+(3) +(4) +(4) +(4) +(4) +(2) +(4)
37 67 275 64 114 465 114
CH CH CH CH CIRR CH CH
Age
601
J. P. Watson et al. TABLE 2
TABLE 3
Hepatitis C virus RNA results from differential ultracentrifugation samples from known hepatitis C virus RNA-positive serum aliquots. NK: not known (no dates of infection were identified for chronic hepatitis C patients). Results in brackets indicate hepatitis C virus RNA was undetectable in high or low density fractions by RT-nested PCR following ultracentrifugation of serum aliquots
Hepatitis C virus quantitative RNA results from serum aliquots of immunocompetent and immunodeticient infected patients analysed
Patient
Weeks postinfection
High density hepatitis C virus RNA
Immunodeficient, acute hepatitis C virus infection 10 + C885 11 + 13 + 12 -ve c935 19 + + 29 61 + 17 C980 -ve 21 (-ve) 54 +
Low density hepatitis C virus RNA -ve + + -ve -ve -ve -ve + (-ve) +
Immunocompetent, acute hepatitis C virus infection + 13 + Cl62 25 + -ve 65 + -ve Immunocompetent, chronic hepatitis C virus infection NK + -ve c35 NK + C8 -ve NK + C356 -ve NK + -ve c5 NK C89 (-ve) (-ve) NK Cl36 (-ve) (-ve) NK + + c37
(C935) presented with low density RNA and then became negative for low density RNA, whilst the two patients with common variable immunodeficiency (CSSS and C980) remained positive for low density RNA in later aliquots. Only l/7 of the chronically infected immunocompetent patients was positive for low density RNA. High density hepatitis C virus RNA was detected early in only 2/4 acute infections, but was detected later in all 4, and was present in all samples from patients with chronic infection in whom hepatitis C virus RNA was detectable following ultracentrifugation (5/7). Hepatitis C virus RNA quantitation in serum Quantitation results for all samples were analysed. Mean hepatitis C virus RNA titres were used in the analysis for the 11 samples in which duplicate results on two aliquots were available, and single results were used in the analysis for the other samples (Table 3). In all, 313 immunodeficient patients and l/8 immunocompetent patients had high hepatitis C virus RNA titres (>6 log,, viral copies/ml). Immunodeficient patients had a significantly higher mean hepati602
Patient
Weeks postinfection
Hepatitis C virus RNA titre (viral copies/ml)
Log,, hepatitis C virus RNA titre (viral copies/ml)
Immunodeficient, acute hepatitis C virus infection 10 3 762 465 C885 11 475 178 13 1 983 12 1567 251 c935 29 1 860 632 61 78 511 17 569 478 C980 21 3 072 139 54 801 922
6.58 5.68 3.30 6.20 6.21 4.89 5.76 6.49 5.90
Immunocompetent, acute hepatitis C virus infection 13 8 940 Cl62 25 213 884
3.95 5.33
Immunocompetent, chronic hepatitis C virus infection 5.11 NK 128 233 c35 5.82 NK 662 543 C8 6.27 NK 1 867 183 C356 5.61 NK 407 481 c5 5.61 NK 406 848 C89 5.78 NK 599 961 Cl36 5.58 NK 316 773 c37
tis C virus RNA titre on presentation (2.0x106f1.3x106 viral copies/ml) than the chronically infected immunocompetent patients (6.4~10% 5.3~10~ viral copies/ml, t=6.22, p.cO.05). The three infected immunodeficient patients were all treated with a-interferon. Patient C885 (common variable immunodeficiency, Fig. 1) was treated from 11-37 weeks post-infection. The hepatitis C virus RNA titre reduced by 2.38 log,, viral copies/ml within 2 weeks of starting treatment and she became hepatitis C virus RNA negative after 4 weeks of treatment. She has remained hepatitis C virus RNA negative with persistently normal liver function tests. Patient C935 (X-linked agammaglobulinaemia, Fig. 3) was treated from 17-26 weeks post-infection, when the treatment was stopped due to unacceptable side effects. He remains hepatitis C virus RNA positive with a lower titre (4.89 log,, viral copies/ml), and is stable with no symptoms and normal liver function tests. He has declined a liver biopsy. Patient C980 (common variable immunodeficiency, Fig. 2) was treated from 1941 weeks post-infection. She did not respond to a-interferon and now has established micronodular cirrhosis on a liver biopsy taken posttreatment (52 weeks post-infection), and has developed ascites. She remains hepatitis C virus RNA positive, with no reduction in hepatitis C virus RNA
HCV density heterogeneity and viral titre 3000
2500
titre = g
2000
si E ._
E 3 1500 5 k E 'E ; 1000
500
1
0 0
20
30
40
50
weeks post-infection
Fig. 1. Patient C885: Acute hepatitis C virus, immunodejcient (common variable immunodejciency}, sponder Alanine transaminase levels and hepatitis C virus RNA titres I&56 weeks post-infection.
a-interferon
re-
7
/9
2500
ti
6
HCV RNA titre 5E
5
2000
+i
$
48
ii ._
$ =.
E z 1500
E 3 'S
5 Z f 'E 2 1000 m
3 2I
500
alanine transaminase
0
IO
1
\
-1
r
0
20
30
weeks post-infection
2
40
50
60&l
Fig. 2. Patient C980: Acute hepatitis C virus, immunodeficient (common variable immunodeficiency), sponder Alanine transaminase levels and hepatitis C virus RNA titres 17-54 weeks post-infection.
a-interferon
non-re-
603
.I. P. Watson et al.
7
3000 HCV RNA titre
5
2500 -1
:
alanine transaminase 1
0
10
20
30
weeks
40
50
60
post-infection
‘“‘If:,,.,,,1
Fig. 3. Patient C935: Acute hepatitis C virus, immunodeficient (X-linked agammaglobulinaemia), spender Alanine transaminase levels and hepatitis C virus RNA titres 12-61 weeks post-infection.
a-interferon
non-re-
7
3000
HCV RNA titre 2500
6
“\ 5s
E
2000
t 5.
ii E ._
4Q $ =
E g 1500
g 3 'S
5 $ t 'E = 1000 m
z 2x
500
1
0
0
0
10
20
weeks 30
post-infection 40
Fig. 4. Patient C162: Acute hepatitis C virus patient, immunocompetent. RNA titres 4-60 weeks post-infection. 604
2
50
6o
Alanine transaminase
levels and hepatitis C virus
HCV density heterogeneity and viral titre
3000 HCV RNA titre
2500
2
2000
$ z ._ i! 5 6 a s ._ ii i?
I
l
15ocl
1000
500
rlanine transaminase
C
c35
C6
C356
C5
C69
Cl36
patient
Fig. 5. Chronic hepatitis C virus patients, immunocompetent.
titre (5.90 log,, viral copies/ml, 54 weeks post-infec-
tion) . An increase in hepatitis C virus RNA detectable in serum was seen following acute infection in the immunocompetent patient (C 162, Fig. 4). Initially, following needle sharing and presentation with an acute hepatitis and a raised alanine transaminase, no hepatitis C virus RNA was detectable in serum 4 weeks post-infection either by reverse transcription polymerase chain reaction assay to the 5’ non-coding region (duplicate samples) or by quantitative hepatitis C virus RNA analysis (duplicate samples). Hepatitis C virus RNA was detectable from 8 weeks postinfection, and the patient has remained RNA positive since then. Very low titres were measured in the first hepatitis C virus RNA positive sample (3.95 log,, copies/ml 8 weeks post-infection), but the titre had increased by 1.38 log,, copies/ml within 3 months (5.33 log,, copies/ml 20 weeks post-infection). The one patient with cirrhosis (C89) in the chronically infected immunocompetent group tested did not have a higher viral titre than the other six patients with chronic hepatitis (Fig. 5).
Discussion In the density heterogeneity analysis, the immunocompetent patient with acute hepatitis and the two
Alanine transaminase
c37
opiLiJ
levels and hepatitis C virus RNA titres.
immunodeficient acute hepatitis patients who did not respond to a-interferon presented with low density hepatitis C virus, and high density hepatitis C virus was only detected later in the course of the infection. This has previously been reported in an acutely infected chimpanzee (5). The immunodeficient ainterferon responder presented with high density hepatitis C virus RNA, but low density hepatitis C virus RNA could also be detected later in the course of her infection. Low density hepatitis C virus is highly infective in chimpanzees irrespective of total hepatitis C virus RNA titre (5), and it has been suggested that this is because low density hepatitis C virus is associated with P-lipoprotein, and the HCV@lipoprotein complex can infect hepatocytes via LDL receptors, using P-lipoprotein as a ligand (7). It may be that this has contributed to the more severe disease seen in the two immunodeficient non-responders. However, it has also been reported that J3-lipoprotein associated low density hepatitis C virus can be found in mild chronic hepatitis C in immunocompetent individuals (4), and it was isolated in l/7 of the immuncompetent chronic hepatitis C virus patients in this study. The nature of the hepatitis C virus RNA found in high density fractions in immunodeficient patients is unclear. Patients with common variable immunodeficiency have defects in cellular and humoral immune 605
J. P. Watson ef al.
responses (14), and patients with X-linked agammaglobulinaemia have a defect in the humoral immune response (15). The common variable immunodeficiency patient who responded to a-interferon and the patient with X-linked agammaglobulinaemia were both ELISA-2 and RIBA- negative, and the other patient with common variable immunodeficiency was ELISA-2 negative and RIBA- indeterminate (~33). Thus the high density fractions detected are unlikely to be immunoglobulin associated, and may be uncoated nucleocapsids, possibly released as a result of lysis of large numbers of infected hepatocytes before complete construction of the hepatitis C virus virion (4), or they could be HDL-associated HCV particles (16). Hepatitis C virus RNA titre was significantly higher in acutely infected immunodeficient patients than in acutely or chronically infected immunocompetent individuals. However, initial total RNA titre did not correlate with clinical course within the group of immiinodeficient patients. The patient who progressed to decompensated cirrhosis presented with the lowest titre of the three immunocompetent patients analysed, and the a-interferon responder presented with the highest titre. Similarly, in the immunocompetent individuals, the one patient with cirrhosis did not have a higher titre than the other patients with chronic hepatitis. It has been shown that the measurement of hepatitis C virus RNA titre alone is not sufficient for predicting in viva infectivity in chimpanzees, and density heterogeneity is a better predictor of infectivity (5). It may be that the same is true in the study of disease progression. Although HCV-RNA titres do fall in the chronic stage of the disease, the absolute RNA titre in the acute stage has no prognostic value. However, the analysis of hepatitis C virus density heterogeneity in serum may yield information of prognostic significance. Our results in the small group of patients analysed in this study are consistent with this hypothesis. Persistent low density hepatitis C virus RNA in the immunodeficient patients was detected in the one patient in whom the disease rapidly progressed, whereas development or persistence of the high density fraction was associated with clearance of the virus in one patient, and low RNA titre and normal liver function tests in another. As outbreaks of acute hepatitis C virus in immunodeficient patients due to contaminated immunoglobulin are fortunately rare, only small numbers of patients can be investigated in studies such as this. However, further investigation of the findings reported in this study are required. Acute non-A non-B hepatitis and acute hepatitis C 606
virus infection in immunodeficient patients have both previously been reported, due to contamination of intravenous immunoglobulin used in the treatment of these patients (15,17-21). We have studied patients infected from a further outbreak. Despite acute infection via the same route of infection (intravenous immunoglobulin infusion), with the same hepatitis C virus strain (genotype la), from the same contaminated batch of intravenous immunoglobulin, the five immunodeficient patients all followed a different clinical course. One patient was hepatitis C virus RNA negative from initial testing, and has had persistently normal liver function tests since then. The patient who died of empyema 20 weeks post-infection had no evidence of hepatitis at post mortem. One patient developed an acute hepatitis, and then rapidly responded to a-interferon with biochemical and virological resolution of disease. Of the two patients who did not respond to a-interferon, one is asymptomatic with persistently normal liver function tests but remains hepatitis C virus RNA positive, and the other has progressed to symptomatic severe liver disease, with decompensated cirrhosis. This variation in host response to viral infection suggests that virus/host interaction is important in the pathogenesis of hepatitis C virus related liver disease.
Acknowledgements We would like to thank Dr Peter Simmonds, University of Edinburgh, for kindly providing genotyping data, Mr Tony McMaster, Freeman Hospital, Newcastle upon Tyne, for expert technical assistance, and Roche Diagnostic Systems for providing the Amplicar HCV Monitor kits used in this study. J.P.W. is supported by a Medical Research Council Training Fellowship.
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14. Spickett GP, Webster ADB, Farrant J. Cellular abnormalities in common variable immunodeficiency. Immunodef Rev 1990; 2: 199-219. 15. Hermaszewski RA, Webster ADB. Primary hypogammaglobulinaemia: a survey of clinical manifestations and complications. QJM 1993; 86: 31-42. 16. Thomssen R, Koch A, Bonk S, Scholz C, Duensing V, Monazahian M. Binding of HCV to LDL, VLDL, HDL, IgG and IgM. Purification of virus protein complexes by anion exchange chromatography and protein A chromatography. Fifth International Symposium on hepatitis C virus end related viruses, 1995; C85. 17. Lever AML, Webster ADB, Brown D, Thomas HC: Non-A Non-B hepatitis occurring in agammaglobulinaemic patients after intravenous immunoglobulin. Lancet 1984; 2: 1062-4. 18. Ochs HD, Fischer SH, Virant FS, Lee ML, Kingdon HS, Wedgewood RJ. Non-A Non-B hepatitis and intravenous immunoglobulin. Lancet 1985; i: 404-5. 19. Bjorklander J, Cunningham-Rundles C, Lundin P, Olsson R, Soderstrom R, Hanson L. Intravenous immunoglobulin prophylaxis causing liver damage in 16 of 77 patients with hypogammaglobulinaemia or IgG subclass deficiency. Am J Med 1988; 84: 107-11. 20. Yap P-L, McOmish F, Webster ADB, Hammarstrom L, Smith CUE, Bjorklander J, Ochs HD, Fischer SH, Quinti I, Simmonds P. Hepatitis C virus transmission by intravenous immunoglobulin. J Hepatol 1994; 21: 455-60. 21. Smith MSH, Webster ADB, Dhillon AP, Dusheiko G, Boulton R, Savage K, Rolles K, Burroughs AK. Orthotopic liver transplantation for chronic hepatitis in two patients with common variable immunodeficiency. Gastroenterology 1995; 108: 879-84.
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Copyright Q European Association for the Study of the Liver 1996 of Hepstology ISSN 0168.8278
Journal
Hepatitis C virus density heterogeneity and viral titre in acute and chronic infection: a comparison of immunodeficient and immunocompetent patients Jonathan P. Watson1’2, Debra J. Bevitt1,2 , Gavin P Spickett3, Geoffrey L. Toms2 and Margaret F. Bassendine’ Departments of ‘Medicine, 2Wrology and 31mmunology. University of Newcastle upon Tyne, Newcastle upon Tyne, UK
Background: Heterogeneities
in the buoyant density of hepatitis C virus RNA have been reported in different groups of patients, and have been attributed to differential binding of viral particles to p-lipoproteins and IgG, and the presence of hepatitis C virus nucleocapsids in circulation. It may be that hepatitis C virus density heterogeneity correlates with the severity of liver disease, hepatitis C virus RNA titre, and the immunocompetence of the patient. Methods and Results: We have analysed five immunodeficient patients (one with hypogammaglobulinaemia and selective IgA deficiency, one with Xlinked agammaglobulinaemia, three with common variable immunodeficiency) who have been acutely infected with the same batch of intravenous immunoglobulin contaminated with hepatitis C virus (genotype la). The course of hepatitis C virus infection in these patients was compared to one immunocompetent patient who presented with acute hepatitis C virus and progressed to chronic disease, and seven immunocompetent patients with chronic hepatitis C. Serum samples were analysed by differential flotation ultracentrifugation in NaCl solution (density 1.063 g/ml). The high and low density fractions were tested for the presence of RNA by RT-PCR. Serum samples were also quantified for
F
acute hepatitis C virus infection, acute hepatitis with jaundice occurs in only 10% of cases (l), and fulminant hepatitis is very rare (1,2). OLLOWING
Received 19 October 1995; revised 22 Febmmy: accepted 23 February 1996
Correspondence: Dr. J. P. Watson, Department of Virology, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Tel.: 0191222 7775. Fax: 0191 222 7775.
hepatitis C virus RNA (Amplicor HCV Monitor kit, Roche Diagnostic Systems). Three quarters of the acutely infected patients analysed presented with low density hepatitis C virus. Low density hepatitis C virus was absent in most chronic infections but persisted in two patients with common variable immunodeficiency. High density hepatitis C virus was detected in the chronic phase in all acutely infected patients in whom the disease persisted, and was present in all samples from PCRpositive patients with chronic infection. Immunodeficient patients had significantly higher hepatitis C virus RNA titres on presentation than immunocompetent patients, but there was no correlation between titre and clinical course of infection. Conclusions: Heterogeneities in the buoyant density of hepatitis C virus RNA have been identified in the patient groups studied. Low density hepatitis C virus is detected more often in acute infection and high density hepatitis C virus is detected more often in chronic infection. Despite acute infection via the same route of infection with the same hepatitis C virus strain, the five immunodeficient patients studied all followed a different clinical course. Key words: Common variable immunodeficiency; Immunoglobulin; P-Lipoprotein; Quantitation.
Most patients follow a subclinical course, and the majority progress to chronic hepatitis (3). The variation in host response and the high rate of viral persistence is poorly understood, and a number of factors including virus genotype, infecting viral dose, selective defects in host cellular and humoral immune responses, and rapid viral sequence evolution may play a role. Hepatitis C virus RNA titre differs in different 599
J. P. Watson et al.
patient groups, and has been reported to be higher in severe disease (4). However, it is unclear whether viral RNA is associated with infectious virus. Hijikata et al. (5) were able to differentiate infectious and non-infectious PCR positive serum by infection of chimpanzees, and reported that hepatitis C virus RNA titre did not correlate with degree of serum infectivity, but hepatitis C virus RNA in infectious serum partitioned at a low density by differential flotation ultracentrifugation. Differences in hepatitis C virus RNA buoyant density between infected individuals have been reported: low density virus has been detected in acute disease and asymptomatic blood donors, and high density virus has been detected in chronic disease (45). The differences in buoyant density have been attributed to differential binding to IgG (high density fraction) and P-lipoprotein (low density fraction) (6,7). The high density fraction has also been attributed to the presence of uncoated nucleocapsids in circulation (4), and this is supported by electron microscopic visualisation of particles of different buoyant densities from hepatitis C virus positive plasma. Immunoelectron microscopy following sucrose density gradient centrifugation of human plasma with high hepatitis C virus RNA titre reveals spherical flavivirus-like particles 55-65 nm in diameter in the low density fractions (1.14-1.16 g/ml) which specifically react with polyclonal and monoclonal anti-HCV envelope antibodies, and smaller core-like particles 30-35 nm in diameter in the high density fractions (1.23-1.27 g/ml) (8). Also, detergent treatment of the low density fraction (1.115 g/ml) extracted from human hepatitis C virus positive serum separated on a potassium bromide density gradient releases icosahedron-shaped particles 33 nm in diameter, which react with anti-HCV core murine monoclonal antibody (9) Direct investigation of the host responses which may drive these changes following acute infection is difficult, as patients are infected with different strains of the virus in varying doses, and duration of infection is often difficult to determine. Recently, a group of patients with a variety of immune deficiencies were infected by a hepatitis C virus contaminated batch of intravenous immunoglobulin (lo), providing an opportunity to study the outcome of a uniform infectious hepatitis C virus challenge in patients with well characterised immune defects. The response of five immunodeficient patients in Newcastle upon Tyne to infection with this contaminated batch has been studied and compared with acutely and chronically infected immunocompetent individuals, by analysis of the clinical course of the disease in each
patient, measurement of hepatitis C virus density heterogeneity following acute infection, and hepatitis C virus RNA quantitation.
Materials and Methods Patients included in the study Five immunodeficient patients were acutely infected during February 1994 with a batch of intravenous immunoglobulin contaminated with hepatitis C virus: three with common variable immunodeficiency, one with X-linked agammaglobulinaemia, and one with hypogammaglobulinaemia and selective IgA deficiency. One patient with common variable immunodeficiency remained hepatitis C virus RNA negative, and the patient with hypogammaglobulinaemia and selective IgA deficiency died of an unrelated illness. Serum samples were collected from the three remaining patients on presentation at the liver clinic and sequentially thereafter (0385: common variable immunodeficiency, serum collected 10-56 weeks post-infection, C980: common variable immunodeficiency, serum collected 17-54 weeks post-infection, C935: X-linked hypogammaglobulinaemia, serum collected 12-61 weeks post-infection). Sequential serum samples were also collected 9-65 weeks postinfection from an immunocompetent patient with acute hepatitis C virus. He presented with acute hepatitis C virus infection following an episode of needle sharing during intravenous drug abuse, and progressed to chronic disease. For comparative study, single serum samples were taken from seven immunocompetent patients with chronic hepatitis C who had not been treated with a-interferon. All samples were stored at -70°C in 100 ~1 aliquots prior to analysis. The mean age of the acute immunodeficient patients was 38.8k8.3 years (2 females, 3 males), the age of the acute immunocompetent patient was 28 years (1 male), and the mean age of the chronic immunocompetent patients was 39.6k5.6 years (7 males). Clinical investigations All patients were treated in the liver clinic at the Freeman Hospital, Newcastle upon Tyne. Sequential liver function tests were performed, and 8/8 immunocompetent and l/3 immunodeficient patients proceeded to liver biopsy for clinical purposes. All patients had hepatitis C virus serology tested by ELISA-2 (Abbott Laboratories, North Chicago, IL, United Biomedical, Hauppage, NY) and RIBA(Ortho Diagnostic Systems). Genotype data was available on all patients (kindly provided by Dr P Simmonds, Edinburgh University).
HCV density heterogeneity and viral titre
Differential$otation ultracentrifigation Serum samples were fractionated by differential flotation ultracentrifugation according to the technique described by Have1 et al. (11). Briefly, 50 pl patient serum was mixed with 8 ml NaCVKBr solution with a density of 1.063 g/ml, and centrifuged at 139,500xg at 14OC for 22 hours. After centrifugation, 1 ml of the low density (top) fraction and 1 ml of the high density (bottom) fraction was collected. The fractions were analysed for the presence of hepatitis C virus RNA by RNA extraction from 100 pl of each fraction, followed by reverse transcription and nested PCR using oligonucleotide primers PTl-4 to amplify part of the hepatitis C virus 5’ non-coding region as previously described (12). Negative controls were used for all reactions, and the guidelines of Kwok & Higuchi (13) were followed to minimise the risk of contamination. Hepatitis C virus RNA quantitation Quantitation of hepatitis C virus RNA in patients’ serum was carried out using the Amplicor HCV Monitor Test (Roche Diagnostic Systems, UK), according to the manufacturer’s instructions. Results were interpreted by selecting the highest dilution of the amplicon that gave an optical density (OD) between 0.5 and 2.0 on the hepatitis C virus microwells (rows A-E) and on the quantitation standard (QS) microwells (rows F-H). The OD for the QS was required to be above 0.5 in at least one well for the determination to be valid. The OD and dilution factors were entered into the HCV monitor.xls Microsoft Excel worksheet file (Roche Diagnostic Systems,
UK, Microsoft Corporation, USA) to compute titre in hepatitis C virus RNA copies/ml.
the
Results Clinical course and investigations Table 1 shows the serological, biochemical, and histological data on the patients who were analysed by differential flotation ultracentrifugation and hepatitis C virus RNA quantitation. Two patients from the study are not included in this table: one patient with common variable immunodeficiency was hepatitis C virus RNA negative with persistently normal liver function tests following intravenous infusion with the contaminated immunoglobulin, and the patient with hypogammaglobulinaemia and selective IgA deficiency died of an unrelated cause (empyema and sepsis) shortly after intravenous infusion with the contaminated batch of immunoglobulin. At post mortem 20 weeks post-infection, no evidence of hepatitis was found in the liver. DifSerentialJZotation ultracentrifigation for analysis of buoyant density of hepatitis C virus particles in vivo Sequential serum samples from the four acutely infected patients (three immunodeficient and one immunocompetent) together with single serum samples from the seven chronically infected patients were analysed by differential flotation ultracentrifugation. Three of the four acutely infected patients presented with low density hepatitis C virus RNA (Table 2). The immunocompetent patient (C162) and the patient with X-linked agammaglobulinaemia
TABLE 1 Clinical and serological details of immunocompetent and immunodeficient hepatitis C virus infected patients analysed. For acutely infected patients the peak alanine transaminase (ah) level is given, followed by the chronic alanine transaminase level at the end of follow up (normal alanine transaminase ~45 iull). Key for liver biopsy data: ND: not done, CH: chronic hepatitis, CIRR: cirrhosis. l*: Genotype 1 but subtype unclassified in this patient Patient
Sex
Genotype
ELISA-2
RIBA-
Alt (m/l)
Liver biopsy
Immunodeficient, acute hepatitis C virus infection F C885 48 M c935 28 F C980 36
la la la
NEG NEG NEG
NEG NEG +(l)C33
2900 to 13 259 to 48 70 to 95
ND ND CIRR
Immunocompetent, Cl62
acute hepatitis C virus infection M 28
1*
POS
+(4)
979 to 98
CH
Immunocompetent, C356 c5 C8 c37 C89 Cl36 c35
chronic hepatitis C virus infection M 43 M 41 M 43 M 29 M 45 M 43 M 33
la la lb lb lb 2b 3
POS POS POS POS POS POS POS
+(3) +(4) +(4) +(4) +(4) +(2) +(4)
37 67 275 64 114 465 114
CH CH CH CH CIRR CH CH
Age
601
J. P. Watson et al. TABLE 2
TABLE 3
Hepatitis C virus RNA results from differential ultracentrifugation samples from known hepatitis C virus RNA-positive serum aliquots. NK: not known (no dates of infection were identified for chronic hepatitis C patients). Results in brackets indicate hepatitis C virus RNA was undetectable in high or low density fractions by RT-nested PCR following ultracentrifugation of serum aliquots
Hepatitis C virus quantitative RNA results from serum aliquots of immunocompetent and immunodeticient infected patients analysed
Patient
Weeks postinfection
High density hepatitis C virus RNA
Immunodeficient, acute hepatitis C virus infection 10 + C885 11 + 13 + 12 -ve c935 19 + + 29 61 + 17 C980 -ve 21 (-ve) 54 +
Low density hepatitis C virus RNA -ve + + -ve -ve -ve -ve + (-ve) +
Immunocompetent, acute hepatitis C virus infection + 13 + Cl62 25 + -ve 65 + -ve Immunocompetent, chronic hepatitis C virus infection NK + -ve c35 NK + C8 -ve NK + C356 -ve NK + -ve c5 NK C89 (-ve) (-ve) NK Cl36 (-ve) (-ve) NK + + c37
(C935) presented with low density RNA and then became negative for low density RNA, whilst the two patients with common variable immunodeficiency (CSSS and C980) remained positive for low density RNA in later aliquots. Only l/7 of the chronically infected immunocompetent patients was positive for low density RNA. High density hepatitis C virus RNA was detected early in only 2/4 acute infections, but was detected later in all 4, and was present in all samples from patients with chronic infection in whom hepatitis C virus RNA was detectable following ultracentrifugation (5/7). Hepatitis C virus RNA quantitation in serum Quantitation results for all samples were analysed. Mean hepatitis C virus RNA titres were used in the analysis for the 11 samples in which duplicate results on two aliquots were available, and single results were used in the analysis for the other samples (Table 3). In all, 313 immunodeficient patients and l/8 immunocompetent patients had high hepatitis C virus RNA titres (>6 log,, viral copies/ml). Immunodeficient patients had a significantly higher mean hepati602
Patient
Weeks postinfection
Hepatitis C virus RNA titre (viral copies/ml)
Log,, hepatitis C virus RNA titre (viral copies/ml)
Immunodeficient, acute hepatitis C virus infection 10 3 762 465 C885 11 475 178 13 1 983 12 1567 251 c935 29 1 860 632 61 78 511 17 569 478 C980 21 3 072 139 54 801 922
6.58 5.68 3.30 6.20 6.21 4.89 5.76 6.49 5.90
Immunocompetent, acute hepatitis C virus infection 13 8 940 Cl62 25 213 884
3.95 5.33
Immunocompetent, chronic hepatitis C virus infection 5.11 NK 128 233 c35 5.82 NK 662 543 C8 6.27 NK 1 867 183 C356 5.61 NK 407 481 c5 5.61 NK 406 848 C89 5.78 NK 599 961 Cl36 5.58 NK 316 773 c37
tis C virus RNA titre on presentation (2.0x106f1.3x106 viral copies/ml) than the chronically infected immunocompetent patients (6.4~10% 5.3~10~ viral copies/ml, t=6.22, p.cO.05). The three infected immunodeficient patients were all treated with a-interferon. Patient C885 (common variable immunodeficiency, Fig. 1) was treated from 11-37 weeks post-infection. The hepatitis C virus RNA titre reduced by 2.38 log,, viral copies/ml within 2 weeks of starting treatment and she became hepatitis C virus RNA negative after 4 weeks of treatment. She has remained hepatitis C virus RNA negative with persistently normal liver function tests. Patient C935 (X-linked agammaglobulinaemia, Fig. 3) was treated from 17-26 weeks post-infection, when the treatment was stopped due to unacceptable side effects. He remains hepatitis C virus RNA positive with a lower titre (4.89 log,, viral copies/ml), and is stable with no symptoms and normal liver function tests. He has declined a liver biopsy. Patient C980 (common variable immunodeficiency, Fig. 2) was treated from 1941 weeks post-infection. She did not respond to a-interferon and now has established micronodular cirrhosis on a liver biopsy taken posttreatment (52 weeks post-infection), and has developed ascites. She remains hepatitis C virus RNA positive, with no reduction in hepatitis C virus RNA
HCV density heterogeneity and viral titre 3000
2500
titre = g
2000
si E ._
E 3 1500 5 k E 'E ; 1000
500
1
0 0
20
30
40
50
weeks post-infection
Fig. 1. Patient C885: Acute hepatitis C virus, immunodejcient (common variable immunodejciency}, sponder Alanine transaminase levels and hepatitis C virus RNA titres I&56 weeks post-infection.
a-interferon
re-
7
/9
2500
ti
6
HCV RNA titre 5E
5
2000
+i
$
48
ii ._
$ =.
E z 1500
E 3 'S
5 Z f 'E 2 1000 m
3 2I
500
alanine transaminase
0
IO
1
\
-1
r
0
20
30
weeks post-infection
2
40
50
60&l
Fig. 2. Patient C980: Acute hepatitis C virus, immunodeficient (common variable immunodeficiency), sponder Alanine transaminase levels and hepatitis C virus RNA titres 17-54 weeks post-infection.
a-interferon
non-re-
603
.I. P. Watson et al.
7
3000 HCV RNA titre
5
2500 -1
:
alanine transaminase 1
0
10
20
30
weeks
40
50
60
post-infection
‘“‘If:,,.,,,1
Fig. 3. Patient C935: Acute hepatitis C virus, immunodeficient (X-linked agammaglobulinaemia), spender Alanine transaminase levels and hepatitis C virus RNA titres 12-61 weeks post-infection.
a-interferon
non-re-
7
3000
HCV RNA titre 2500
6
“\ 5s
E
2000
t 5.
ii E ._
4Q $ =
E g 1500
g 3 'S
5 $ t 'E = 1000 m
z 2x
500
1
0
0
0
10
20
weeks 30
post-infection 40
Fig. 4. Patient C162: Acute hepatitis C virus patient, immunocompetent. RNA titres 4-60 weeks post-infection. 604
2
50
6o
Alanine transaminase
levels and hepatitis C virus
HCV density heterogeneity and viral titre
3000 HCV RNA titre
2500
2
2000
$ z ._ i! 5 6 a s ._ ii i?
I
l
15ocl
1000
500
rlanine transaminase
C
c35
C6
C356
C5
C69
Cl36
patient
Fig. 5. Chronic hepatitis C virus patients, immunocompetent.
titre (5.90 log,, viral copies/ml, 54 weeks post-infec-
tion) . An increase in hepatitis C virus RNA detectable in serum was seen following acute infection in the immunocompetent patient (C 162, Fig. 4). Initially, following needle sharing and presentation with an acute hepatitis and a raised alanine transaminase, no hepatitis C virus RNA was detectable in serum 4 weeks post-infection either by reverse transcription polymerase chain reaction assay to the 5’ non-coding region (duplicate samples) or by quantitative hepatitis C virus RNA analysis (duplicate samples). Hepatitis C virus RNA was detectable from 8 weeks postinfection, and the patient has remained RNA positive since then. Very low titres were measured in the first hepatitis C virus RNA positive sample (3.95 log,, copies/ml 8 weeks post-infection), but the titre had increased by 1.38 log,, copies/ml within 3 months (5.33 log,, copies/ml 20 weeks post-infection). The one patient with cirrhosis (C89) in the chronically infected immunocompetent group tested did not have a higher viral titre than the other six patients with chronic hepatitis (Fig. 5).
Discussion In the density heterogeneity analysis, the immunocompetent patient with acute hepatitis and the two
Alanine transaminase
c37
opiLiJ
levels and hepatitis C virus RNA titres.
immunodeficient acute hepatitis patients who did not respond to a-interferon presented with low density hepatitis C virus, and high density hepatitis C virus was only detected later in the course of the infection. This has previously been reported in an acutely infected chimpanzee (5). The immunodeficient ainterferon responder presented with high density hepatitis C virus RNA, but low density hepatitis C virus RNA could also be detected later in the course of her infection. Low density hepatitis C virus is highly infective in chimpanzees irrespective of total hepatitis C virus RNA titre (5), and it has been suggested that this is because low density hepatitis C virus is associated with P-lipoprotein, and the HCV@lipoprotein complex can infect hepatocytes via LDL receptors, using P-lipoprotein as a ligand (7). It may be that this has contributed to the more severe disease seen in the two immunodeficient non-responders. However, it has also been reported that J3-lipoprotein associated low density hepatitis C virus can be found in mild chronic hepatitis C in immunocompetent individuals (4), and it was isolated in l/7 of the immuncompetent chronic hepatitis C virus patients in this study. The nature of the hepatitis C virus RNA found in high density fractions in immunodeficient patients is unclear. Patients with common variable immunodeficiency have defects in cellular and humoral immune 605
J. P. Watson ef al.
responses (14), and patients with X-linked agammaglobulinaemia have a defect in the humoral immune response (15). The common variable immunodeficiency patient who responded to a-interferon and the patient with X-linked agammaglobulinaemia were both ELISA-2 and RIBA- negative, and the other patient with common variable immunodeficiency was ELISA-2 negative and RIBA- indeterminate (~33). Thus the high density fractions detected are unlikely to be immunoglobulin associated, and may be uncoated nucleocapsids, possibly released as a result of lysis of large numbers of infected hepatocytes before complete construction of the hepatitis C virus virion (4), or they could be HDL-associated HCV particles (16). Hepatitis C virus RNA titre was significantly higher in acutely infected immunodeficient patients than in acutely or chronically infected immunocompetent individuals. However, initial total RNA titre did not correlate with clinical course within the group of immiinodeficient patients. The patient who progressed to decompensated cirrhosis presented with the lowest titre of the three immunocompetent patients analysed, and the a-interferon responder presented with the highest titre. Similarly, in the immunocompetent individuals, the one patient with cirrhosis did not have a higher titre than the other patients with chronic hepatitis. It has been shown that the measurement of hepatitis C virus RNA titre alone is not sufficient for predicting in viva infectivity in chimpanzees, and density heterogeneity is a better predictor of infectivity (5). It may be that the same is true in the study of disease progression. Although HCV-RNA titres do fall in the chronic stage of the disease, the absolute RNA titre in the acute stage has no prognostic value. However, the analysis of hepatitis C virus density heterogeneity in serum may yield information of prognostic significance. Our results in the small group of patients analysed in this study are consistent with this hypothesis. Persistent low density hepatitis C virus RNA in the immunodeficient patients was detected in the one patient in whom the disease rapidly progressed, whereas development or persistence of the high density fraction was associated with clearance of the virus in one patient, and low RNA titre and normal liver function tests in another. As outbreaks of acute hepatitis C virus in immunodeficient patients due to contaminated immunoglobulin are fortunately rare, only small numbers of patients can be investigated in studies such as this. However, further investigation of the findings reported in this study are required. Acute non-A non-B hepatitis and acute hepatitis C 606
virus infection in immunodeficient patients have both previously been reported, due to contamination of intravenous immunoglobulin used in the treatment of these patients (15,17-21). We have studied patients infected from a further outbreak. Despite acute infection via the same route of infection (intravenous immunoglobulin infusion), with the same hepatitis C virus strain (genotype la), from the same contaminated batch of intravenous immunoglobulin, the five immunodeficient patients all followed a different clinical course. One patient was hepatitis C virus RNA negative from initial testing, and has had persistently normal liver function tests since then. The patient who died of empyema 20 weeks post-infection had no evidence of hepatitis at post mortem. One patient developed an acute hepatitis, and then rapidly responded to a-interferon with biochemical and virological resolution of disease. Of the two patients who did not respond to a-interferon, one is asymptomatic with persistently normal liver function tests but remains hepatitis C virus RNA positive, and the other has progressed to symptomatic severe liver disease, with decompensated cirrhosis. This variation in host response to viral infection suggests that virus/host interaction is important in the pathogenesis of hepatitis C virus related liver disease.
Acknowledgements We would like to thank Dr Peter Simmonds, University of Edinburgh, for kindly providing genotyping data, Mr Tony McMaster, Freeman Hospital, Newcastle upon Tyne, for expert technical assistance, and Roche Diagnostic Systems for providing the Amplicar HCV Monitor kits used in this study. J.P.W. is supported by a Medical Research Council Training Fellowship.
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