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RAPID COMMUNICATION
Increasing Hepatitis C Virus RNA Levels in Hemophiliacs: Relationship to
Human Immunodeficiency Virus Infection and Liver Disease
By M. Elaine Eyster, Michael W. Fried, Adrian M. Di Bisceglie, and James J. Goedert
for the Multicenter Hemophilia Cohort Study
We have previously observed an increased frequency
of liver
failure in human immunodeficiency virus (HlV)-infected
hemophiliacs. The purpose of this study was t o quantitate
hepatitis C virus (HCV) RNA levels in serial samples from
HIV-seropositive (HIV') and HIV-seronegative (HIV-) hemophiliacs before and after HIV seroconversion, and
t o examine
the relationship of HCV RNA levels t o CD4 cell counts and
t o hepatic dysfunction over time.HCV RNAlevels were measured on serial samples of serum stored frozen from 17
HCV+/HIV' and 17 HCV'/HIV- subjects matched within 5
years of their birth dates. All were HCV' before study entry.
HCV RNA levels were quantitated by a branched DNA-enhanced label amplification (bDNA) assay. For samples less
than the cut off, HCVRNA wes measured by tho nested
polymerase chain reaction. Individual changes over time,
clinical groups, and mean values
within predeterminedtime
windows were comp8red with Wilcoxon rank sum tests.
Mean HCV RNA levels increasedfrom 2.76 (standard error
[SEI 1.33) x 10' t o 2.84(SE1.39) x 10" eq/mL during the
first 2 years after HIV seroconversion ( P = .006). Baseline
HCV RNA levels in the praHlVseroconvmion group were
not significantly different from the baseline levelsin those
who remained HIV-(P = .79). Over the entire period of study,
HCVRNA levels increased nearly threefold in those who
remained HIV- (mean 9.47 [SE 4.781 x 10' t o 2.81 [SE 1.131
x lO'/mL; P = .02). Among those who became HIV',HCV
RNA levels increased%-fold ( m m 2.85 [SE 1.261 x 105 t o
1.66 [SE 0.571 x lo7eq/mL; P = .OOOl). The rate ofincrease
in HCV RNA levels was eightfold faster for HIV' subjects
than for subjects who remained HIV- (P = .009). HCV RNA
levels increased
twofold higher in 5 subjects who developed
liver failure compared with the 12 who did not (P = .U).
HCV RNA levels corrolated signifkantly with CD4 counts (R
= -.33, P = .01) and serum aspartate arninotransfmraselevels (AST) (R = .36, P = .007). We concludo that HCV RNA
levels are significantly higher in HN' than in HIV- multitransfused hemophiliacs. HCV load increases over time, is
enhanced by HIV, and further increasesas immune deficiency progresses. HCV RNA levels are directly associated
with high AST lovels. These findings suggest that HIV-induced immune deficiency may promote increasedHCV replication.
6 1994 by The American Society of Hematology.
T
of liver failure. These findings suggest that HIV or its associated immune deficiency state may accelerate the development of liver failure, perhaps by enhancing HCV replication.
The purpose of this study was to compare serial measurements of HCV RNA in HIV- and HIV+ hemophiliacs before
and after HIV seroconversion, and to examine the relationship of HCVRNA levels to CD4 counts and to hepatic
dysfunction over time.
HE VAST MAJORITY of persons with hemophilia who
have been transfused with non-heat-treated clotting
factor concentrates have been infected with hepatitis C virus
(HCV).'" Most have also been infected with the human immunodeficiency virus (HIV).~
We have previously reported that liver failure occurs more
frequently in HCV-seropositive (HCV+) hemophiliacs who
are HIV-seropositive ( H I V ) than in those who are HIVseronegative (HIV-).' Those with lower T4cell (CD4) lymphocyte counts or lymphocytopenia have an increased risk
From theDivision of Hematology, Department of Medicine, Pennsylvania State University College of Medicine, Hershey, PA; the
Liver Diseases Section, National Institute of Diabetes and Digestive
and Kidney Diseases, Bethesda, MD; and the Viral Epidemiology
Branch, National Cancer Institute, Rockville, MD.
Submitted February 7, 1994; accepted May 24, 1994.
Supported in part byContract No. SP 173656from the Pennsylvania Department of Health, gifrs from the Brandywine Valley Hemophilia Foundation and the Alice Livingston Trout Family Fund, and
by National Cancer Institute Contracts NOl-CP-85649 and NOlCP-33002-21.
Presented at the 35th Annual Meeting of the American Socicty of
Hematology, St Louis. MO, December 7, 1993.
Address reprint requests to M.Elaine Eyster, MD, Division of
Hematology, Department of Medicine, Pennsylvania State University
College of Medicine, PO Box 850, Hershey, PA 17033.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
6 1994 by The American Society of Hematology.
ooW-4971/94/8404-0049$3.00/0
1020
PATIENTS AND METHODS
The patients for this study were a subset of a well-characterized
cohort of 223 persons with hemophilia with known HIV seroconversion dates and HCVstatus who were observed regularly in a comprehensive care clinic since 1973 and enrolled with informed consent
in the Multicenter Hemophilia Cohort Study initiated in1982.'.*
Periodic evaluations and testing consisting ofa complete blood
count, serum aspartate aminotransferase (AST), hepatitis B surface
antigen (HBsAg), antibody (antiHBs), and T4 lymphocyte (CM)
counts had been performed at intervals of 6 months to 1 year as
previously
Tests for HCV antibody were initiated in
1989 with the enzyme-linked immunosorbent assay (ELISA; Ortho
Diagnostics System, Raritan, NJ). Aliquots of these sera stored frozen were retested using a second generation recombinant immunoblot assay (RIBA-2; Chiron Corp. Emecyville, CA). Samples were
considered positive if they reacted with 2 or more of the 4 test
antigens.'
From the full cohort, we identified 25 individuals without HBsAg
who were HCV+ HIV' and who had sufticient sera stored at -20°C
to -70°C for testing of HCV RNA before HIV seroconversion, 2
years or less after seroconversion, and 5 years or more after HIV
seroconversion. For this investigation, we included all 17 HCV'
HIV' individuals for whomwe could identify HCV' controls
matched within 5 years of their birth dates who were seronegative
for HBsAg and for H N ; were intensively exposed to nonscreened,
Blood, VOI 84, NO 4 (August 15). 1994: pp 1020-1023
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1021
HCV RNA LEVELS IN HEMOPHILIACS
non-heat-treated blood products; and had at least 3 serum specimens
stored frozen over comparable periods of time. After the 17 H C V
HIV+ and 17 HCV+ HIV- subjects were identified, we tested interval
sera on those with liver failure. Liver failure was defined as bleeding
esophageal varices, ascites, hepatic encephalopathy, or persistent
hyperbilirubinemia (>2 months) not attributable to other known
causes, as previously de~cribed.~
Dates of study entry were determined by the first available specimen that was found to be HCV'
by RIBA-2. No subjects had received interferon a as of December
31, 1992, when data were censored.
HCV RNA levels were quantified by a branched chain DNAenhanced label amplification (bDNA) assay (Chiron). In this assay,
nucleic acid from serum samples is specifically hybridized to synthetic oligonucleotides and captured onto the surface of a 96-well
microplate. Multiple copies of a synthetic branched DNA molecule
and a conjugated alkaline phosphatase-labeled probe are then annealed to the immobilized complex (signal amplification) and detected with a chemiluminescent substrate. Because the target is not
amplified, the signal is proportional to the physiologic level of target
nucleic acid, and the quantity of nucleic acid in the sample can be
accurately measured. For samples with values less than the cut-off
point of 3.5 X 10' genome equivalents/mL, HCV was measured by
the polymerase chain reaction (PCR), using nested primers from the
5' noncoding region of the HCV genome as previously described.''
For quantification estimates and statistical comparisons, PCR-positive samples were assigned a value of 1.75 X lo" eq/mL (one-half the
cut-off value for positivity by the bDNA assay), and PCR-negative
samples were assigned a value of 0. Mean values and standard errors
(SE) were calculated within predetermined time windows. Change
in HCV RNA level per unit time for each subject was estimated as
the slope between the first and the last specimen. Individual changes
over time and between groups were compared with Wilcoxon rank
sum tests. Spearman rank order correlations were calculated for
simultaneous HCV RNA level, AST level, and CD4 lymphocyte
count.
RESULTS
The 17 HCV+ HIV+ subjects had a mean age of 28.6 ?
3.9 (SE) years (median, 26; range, 3 to 62), and the 17 HCV'
HIV- subjects had a mean age of 30.5 ? 4.1 years (median,
27; range, 6 to 64) at the time their baseline assays were
performed (median year, 1981 for HIV+ and 1982 for HIVsubjects). HIV+ subjects were observed for a mean of 9.2
-1- 0.68 and a median of 9.6 years (range, 4.3 to 14.4 years).
HIV- subjects were observed for a mean of 8.9 2 0.68 and
a median of 9.2 years (range, 4.3 to 13 years).
Of the HCV+ HIV+ subjects who were testedpre-HIV
seroconversion, 7 were HCV RNA- by PCR, 6 were PCR+
but bDNA-, and 3 were bDNA+. The pre-HIV seroconversion sample of 1 subject was unsuitable for testing. Mean
HCV RNA levels increased from 2.76 (SE 1.33) X lo5 to
2.84 (SE 1.39) X lo6 eq/mL. during the first 2 years after
HIV seroconversion ( P = .006; Fig 1). After the initial increase, HCV RNA levels remained stable during the 2 to 5
years after HIV seroconversion (1.48 [SE 0.561 X lo6 eq/
mL). However, by 5 to 13 years postseroconversion, the
HCV RNA mean level had increased markedly to 2.24 (SE
0.84) X 107/mL( P = .05).
Baseline HCV RNA levels in the pre-HIV seroconversion
samples were not significantly different from the baseline
levels in those who remained HIV- ( P = .79). Over the
entire period of the study, HCV RNA levels increased nearly
Pre HIV
Seroconversion
lo*
Post HIV
Seroconversion
7
?
107
-E
>P
0
I
106
3
105
* PCR positive
0 = # of patients
t PCR negative
Excludes one with specimen
unsuitable for testing
Fig 1. HCV RNA levels pre-HIV seroconversionand within 2 years
post-HIV seroconversion in 16 subjects. Preseroconversion, 13 subjects were below the detection limit of 3.5 x I O 5 eqlmL for the bDNA
assay. Postseroconversion,5 subjects remained below the detection
limit. Four of the five were positive for HCV RNA by PCR. Three of
the four PCR+ subjects were PCR- before HIV seroconversion.
threefold in those who remained HIV- (mean, 9.47 [SE 4.781
X lo5 to2.81 [SE 1.131 X lo6 eq/mL; P = .02; Fig 2A).
Among those who became HIV+, HCVRNA levels increased %-fold (mean 2.85 [SE 1.261 X lo5 to 1.66 [SE
0.571 X lo7 eq/mL; P = .0001; Fig 2B). The rate of increase
in HCV RNA levels was eightfold faster for H I V subjects
(mean slope, 1.58 X lo6 eq/mL per year) than for subjects
who remained HIV- (mean slope, 1.90 X lo5 eq/mL per
year; P = .009). HCV RNA levels increased twofold faster
in the 5 subjects who developed liver failure compared with
the 12 who did not(mean slope, 2.44 X lo6 v 1.22 X 10'
eq/mL per year; P = .43).
There was a significant correlation between HCV RNA
levels and CD4 counts obtained on the 56 dates when both
were measured (Spearman R = -.33, P = .01). This correlation was entirely driven by values on 23 dates that were at
least 2 years post-HIV seroconversion (R= -.56, P = .006).
AST levels also were significantly correlated withHCV
RNA levels ( R = .36, P = .007) for 53 samples drawn on
the same dates as those for HCV RNA levels.
DISCUSSION
Virtually all persons with hemophilia who weretransfused
with clotting factor concentrates before the implementation
of viral inactivation procedures in 1984 became infected
with HCV,"5.7 and
the majority remain chronically infected."
Serum levels of HCV RNA are variably associated with the
degree of biochemical and histologic liver inflammati~n,'~.'~
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EYSTER
1022
HCV+ / HIV109
,
ETAL
HCV+ / HIV+
lB
1
108
107
106
3.5
105
Fig 2. Serial HCV RNA levels on 17 HCV+/HIV- subjects (AI and 17 HCV'/HIV+ subjects (B).
and chronic active hepatitis or cirrhosis has been reported
in 10% to 20% of multitransfused adults with hemophilia
who underwent liver biopsies or whohad post mortem examination~."~'~
In our cohort of hemophiliacs in Central Pennsylvania,
we have previously reported that 8 (9%) of 91 HCV' HIV'
acquired immunodeficiency syndrome (AIDS)-free persons
compared with none of 58 HCV- HIV+ persons developed
liver f a i l ~ r e In
. ~ the present study, weused a quantitative
branched DNA-enhanced label amplification assay to compare serial measurements of HCV RNA in a subcohort of
age-matched H I T and HIV' hemophiliacs. Mean baseline
levels of HCV RNA in the two groups were similar. Mean
HCV levels increased significantly over the next 5 to 12 or
more years in both groups, but the increase was eightfold
greater in the HIV+ than in the HIV- group during a period
of time when viral inactivated concentrates were in widespread use. HCV RNA levels increased twice as fast in those
HCV' HIV+ individuals who developed liver failure, compared with those who did not, but the sample size was too
small to reach significance. One with liver failure had micronodular cirrhosis; another had extensive cholestasis (data
not presented). We have not yet observed a case of liver
failure during 167 person years of observation in our HCV'
HIV- patients. HCV RNA levels were positively correlated
with AST levels in both HIV+ and HIV- patients.
A strong negative correlation was shown between HCV
RNA levels and CD4 counts, suggesting that HIV-induced
immune deficiency may allow increased HCV replication.
Very high viral loads of 2 X lo7 to 3 X 108/mLwere seen
only in HIV+ individuals. Thus, HCV may be reactivated or
inefficiently cleared in the immune deficient host. Alterna-
tively, immune deficient individuals may be less able to
respond to the emergence of certain HCV variants, or may
be more susceptible to reinfection from plasma concentrates
containing virus below the level of detection. In any case,
there is good reason to suspect that HIV' hemophiliacs are
at higher risk for the development of HCV-related liver failure as well as for the transmission of HCV to their sexual
partners.18
The increase in HCV RNA levels over several years even
in HIV- individuals is of great interest. This suggests that
HCV propagates more efficiently over time, implying that
the immune system is not adequate to control the infection.
T-cell deviations including lower CD4+ percentages and
counts and CD56+ natural killer subsets have been previously reported in HIV- persons with hemophilia."
The pathogenesis of hepatocellulardamage byHCV is
poorly understood. As withchronic hepatitis B infection, evidence is emerging that liverdamage may be mediated by the
immune reaction to infected hepatocytes, rather than by the
virus itself." However, the demonstration of HCV antigens
in liver biopsies and the correlation between
levels of viremia
and degree of lobular inflammation suggests that HCV may
be directly cytopathic to liver cell^.'^.'^"' Our data provide
strong circumstantial evidence that cellular immunity is important in controlling HCV infection, and that HIV-induced
immune deficiency may permit increased HCV replication.
Confirmation of thepossibleassociation of highlevels of
HCV RNA with more severe liver
disease will require analysis
of data from our larger multicenter cohort study. However,
our findingsfavor the mechanismof direct cytopathicity rather
than cellular immune reactivityas the cause of hepatocellular
damage in persons with chronic HCV infection.
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HCVRNA LEVELS IN HEMOPHILIACS
Recently, Sherman et alZ2have also reported that HCV
RNA levels measured by the same methodology were higher
in 13 HIV+ than in 30 H I T individuals. They did not find
a correlation between HCVRNA levels (mean, 3.8 X lo7
in HIV+ v 1.18 X lo7 in HIV- patients) and CD4 counts
(range, 292 to 1,024 cells/pL). However, this is not surprising because our strong negative correlation was driven by
lower CD4 counts (median, 201; data not shown) more than
2 years after HIV seroconversion.
Using the same quantitative bDNA assay, Lau et a l l 3 have
reported median HCV RNA levels of 2.70 X lo6 eq/mL in
recipients of blood transfusions and 6.3 X lo5 eq/mL in
health care workers and intravenous drug users who were
participating in clinical trials with interferon a. Seven of 13
subjects (54%) who were bDNA- but W R + had a sustained
response to interferon a , compared with only 4 of 33 who
were bDNA'. Fourteen of17of
our HIV- subjects were
bDNA+, and mean levels of lo7 eq/mL were observed in
our H I V subjects. These observations suggest that sustained
remissions with interferon a may be difficult to achieve in
HCV' hemophiliacs, particularly those who are HIV+.
In summary, we have shown that HCV RNA levels are
significantly higher in HIV+ than in HIV- multitransfused
hemophiliacs. HCV load increases over time, is enhanced by
HIV, and further increases as immune deficiency progresses.
HCV RNA levels are strongly associated with AST levels.
Some HCV+ HIV+ hemophiliacs with rapidly progressive
liver failure have very high levels of HCV viremia. Our
findings emphasize the need for clinical trials to assess the
benefit of interferon a in HCV+/HIV+ as well as HCV+/
HIV- hemophiliacs. This should now be possible using
quantitative HCV RNA assays to assess response to therapy.
ACKNOWLEDGMENT
The authors thank Susan Wilson (Atlantic Research Corp) for
computer programming, and the investigators from 16 institutions
comprising the Multicenter Hemophilia Cohort study listed in Eyster
et al' for their helpful comments.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1994 84: 1020-1023
Increasing hepatitis C virus RNA levels in hemophiliacs: relationship
to human immunodeficiency virus infection and liver disease.
Multicenter Hemophilia Cohort Study
ME Eyster, MW Fried, AM Di Bisceglie and JJ Goedert
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