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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4333-4336
TT Virus Is Present in a High Frequency of Italian Hemophilic Patients
Transfused With Plasma-Derived Clotting Factor Concentrates
By
Benjamin P. Chen,
Maria Grazia Rumi,
Massimo Colombo,
Yu-Huei Lin,
Latha Ramaswamy,
Jac Luna,
Jen-Kuei Liu,
Daniele Prati, and
Pier
Mannuccio Mannucci
From Sentinel BioSciences, Inc, Palo Alto, CA; the Department of
Internal Medicine, Blood Transfusion and Transplant Immunology Center,
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore
Hospital, University of Milan, Milan, Italy.
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ABSTRACT |
The prevalence of the blood-borne TT virus (TTV) in Italian
hemophiliacs treated with different preparations of factor VIII was
determined. Of the 178 hemophilic patients (mean age, 29 years), TTV-DNA was found in 123 (69%), in comparison to 22 of 100 (22%) blood donors (P < .0001). Of the 123 patients who tested
positive for TTV, significant numbers were also infected with human
hepatitis viruses and/or human immunodeficiency virus (HIV): 31% had
TTV and hepatitis C virus (HCV), 22% had TTV, and at
least 2 of the 4 known human blood-borne viruses tested, whereas 15%
had TTV alone. The risk of acquiring TTV alone was only slightly higher in recipients of unmodified plasma factor concentrates (78%, odds ratio, 1.24; 95% confidence interval [CI], 0.27 to
5.79) than in patients treated with virus inactivated concentrates
(67%), whereas the risk was significantly lower in recipients of
recombinant factors (11%, odds ratio, 0.09; 95% CI, 0.01 to 0.52).
Serum alanine aminotransferase (ALT) levels were elevated in 2 of 27 patients (7%) with TTV alone compared with 43 of 56 patients (77%)
coinfected with TTV and HCV and compared with 16 of 21 patients (76%)
with HCV alone. Taken together, these results indicate that TTV
frequently infects Italian hemophiliacs treated with plasma-derived
factor VIII concentrates, both unmodified and virus-inactivated. Our results do not suggest a causal effect of TTV on chronic liver disease
in these patients.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMOPHILIC PATIENTS ARE at high risk of
acquiring viral hepatitis after repeated treatment with plasma-derived
clotting factor products.1-3 With the implementation of
procedures for inactivating viruses in plasma-derived products, a
dramatic decrease in the transmission of hepatitis B and C viruses has
been observed in this patient population.4,5 Virus
inactivation procedures also have been successful in reducing the risk
of transmitting the blood-borne hepatitis G virus (HGV).6,7
However, other blood-borne viruses, known and as yet unidentified,
might escape the current virucidal procedures in plasma products and
establish infections in the recipients. For instance, an outbreak of
hepatitis A virus (HAV) infection occurred in 1995 among European
patients infused with factor VIII (FVIII) preparations treated with
solvent-detergent, a procedure ineffective against a nonenveloped virus
such as HAV.8
Recently, a new nonenveloped, blood-borne, single-stranded DNA virus,
TTV, was identified in sera of high-risk patients using a molecular
subtraction technique.9,10 This virus was subsequently detected in blood donors worldwide11,12 and in higher
frequencies in recipients of repeated transfusions of blood
products.13-19 The detection of the TTV-DNA in patient sera
in association with elevated levels of alanine aminotransferase (ALT)
raised the possibility that TTV may be a new hepatotropic
virus.9,17 Recent studies have shown that TTV can be
detected in blood products and in multitransfused hemophilic patients,
and it remains controversial if virucidal procedures are effective
against TTV.14-16 Therefore, this study was initiated to
assess the prevalence and potential hepatotoxicity of TTV in Italian
hemophiliacs treated with different clotting factor preparations:
unmodified plasma-derived clotting factor concentrate, concentrates
treated with virucidal procedures, or recombinant FVIII.
| |
MATERIALS AND METHODS |
Study subjects.
A total of 178 consecutive Italian hemophilic patients (mean age, 29 years; range, 2 to 73 years) treated at the Angelo Bianchi Bonomi
Hemophilia and Thrombosis Center, Milano, Italy, were included in this
study. The only criterion used for selection was that all patients had
been prospectively followed with annual liver function tests since
their first treatment at the Center (mean follow-up of 16 years; range, 2 to 27 years). The epidemiological and clinical
characteristics of these patients are summarized in
Table 1. The 38 patients with mild
hemophilia had been infused with concentrates because they did not
respond to 1-deamino-8-D-arginine vasopressin (DDAVP). One hundred
twenty-seven (71%) patients had been treated with unmodified
concentrates until 1985 and with virus-inactivated concentrates
thereafter; 33 (19%) patients were given only virus-inactivated
concentrates, and 18 (10%) were given recombinant FVIII preparations
exclusively. The virus inactivation method implemented by manufacturers
from 1984 to 1987 was dry-heating at 60° to 68°C; this
procedure was effective against the transmission of human
immunodeficiency virus (HIV), but not of hepatitis C virus
(HCV).20 Since 1987, more effective virucidal procedures, such as dry-heating at >80°C, vapor heating, solvent/detergent, and pasteurization were adopted. Although most patients received more
than 1 virally-inactivated product, the solvent/detergent method was
the most widely used, with at least two thirds of the patients having
been treated with concentrates inactivated with this method. One
hundred volunteer blood donors (73 men, 27 women; mean age, 36 years)
from the hospital blood bank, all with persistently normal ALT and no
serum hepatitis B surface antigen (HBsAg), anti-HCV, or
anti-HIV were included as controls.
Virological and biochemical measurements in patient sera.
Serum ALT activity was measured by an automated standardized
colorimetric assay (normal value,  40 U/L). HBsAg and antibodies to
HIV (anti-HIV) in sera were tested by immunoenzymatic assays (Abbott
Laboratories, North Chicago, IL). Serum HGV-RNA was detected by
polymerase chain reaction (PCR) as previously described,7 and HCV-RNA was detected by a nested PCR using primers of the 5'
noncoding region of the viral genome as previously
described.21
Determination of TTV DNA by PCR.
Serum TTV DNA was detected by a seminested PCR as previously
described.10,13 Specifically, 7 µL of serum DNA (the
amount of DNA purified from an equivalent of 50 µL of serum) was
amplified by PCR in a 9600 thermal cycler (Perkin-Elmer, Emeryville,
CA). The following PCR protocol was used: 1 cycle at 95°C for 9 minutes, 35 cycles at 94°C for 30 seconds, 58°C for 30 seconds,
72°C for 45 seconds, and 1 cycle at 72°C for 7 minutes. The
reaction conditions were 30 pmoles of each sense (N059 5'-ACA GAC
AGA GGA GAA GGC AAC ATG-3') and antisense (NG063 5'-CTG GCA
TTT TAC CAT TTC CAA AGT-3') primers with 2.5 U of AmpliTaq Gold
(Perkin-Elmer) in 50 µL of reaction volume. Five microliters of the
amplification products was submitted to a second round of PCR using the
same reaction mix and conditions as above with a seminested primer set
(sense NG061 5' GGC AAC ATG TTA TGG ATA GAC TGG 3',
antisense NG063). Multiple positive and negative controls were included in each PCR assay, and PCR products were analyzed in a 2% agarose gel
electrophoresis with ethidium bromide staining. Results of all samples
tested positive for TTV DNA in the PCR were confirmed in separate
assays and PCR products were also sequence-confirmed by automated
sequencing on an ABI 373 sequencer (Perkin Elmer, Foster City, CA).
Statistical analysis.
The prevalence of serum TTV-DNA and other virus markers was compared by
the  2 test and/or Fisher's exact test. Differences in
the prevalence of serum TTV-DNA and other virus markers, on the basis
of factor VIII preparations, were estimated by the odds ratio and their approximate 95% confidence interval (CI).
| |
RESULTS |
TTV-DNA was detected in the serum of 123 hemophilic patients and 22 blood donors (69% v 22%, P < .0001). The prevalence
of TTV-DNA was higher in the 127 recipients of unmodified concentrates (78%) and in the 33 recipients of virus-inactivated products (67%) than in the 18 recipients of recombinant concentrates only (11%) (Table 2). To assess whether a correlation
exist between treatment intensity and risk of acquiring TTV, TTV-DNA
seroprevalences were related to the severity of hemophilia. TTV-DNA was
present in 102 of 140 patients (73%) with severe or moderate
hemophilia (FVIII 5% or less) compared with 21 of 38 (55%) with mild
hemophilia (P = .053).
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Table 2.
Prevalence of Viral Markers in Sera of Blood Donors and
Hemophilic Patients Treated With Different Concentrates
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HBsAg was detected in 6 (5%) recipients of unmodified concentrates
only. The prevalence of HCV-RNA and anti-HIV was significantly greater
in patients treated with unmodified concentrates (85% and 24%,
respectively) than in the recipients of virus-inactivated (15% and 0)
or recombinant concentrates (0 and 0). In contrast, HGV-RNA was rarely
detected in the recipients of both unmodified concentrates and
virus-inactivated or recombinant factors (8% v 3% and 0, respectively) (Table 2). TTV-DNA alone was detected in 27 patients
(15%), HCV-RNA alone in 21 (12%), TTV-DNA+HCV-RNA in 56 (31%), more
than 1 virus in 42 (24%), and no virus in 32 (18%)
(Table 3).
Overall, 98 patients (55%) had either intermittently or persistently
elevated serum ALT values. Only 2 of 27 (7%) patients exclusively
infected by TTV had abnormal ALT compared with 43 of the 56 (77%) who
were coinfected by TTV and HCV, and the 16 of 21 (76%) who circulated
HCV-RNA alone (Table 3). The risk of acquiring TTV alone was 1.24 (95%
CI, 0.27 to 5.79) for the recipients of unmodified concentrates and
0.09 (95% CI, 0.01 to 0.52) for the recipients of recombinant factors
compared with patients with virus-inactivated concentrates. The
corresponding figures for the risk of acquiring any other virus were
41.4 (95% CI, 9.3 to 204.5) and 0 (95% CI, 0 to 0.82), respectively
(Table 4).
To assess whether exposure to TTV was affected by patients' age,
TTV-DNA prevalences were calculated in donors and patients after
stratification by decades (Table 5). In
donors, TTV-DNA rates were 19% in the 31 subjects aged 20 to 30 years,
16% in the 31 subjects aged 31 to 40 years, 28% in the 21 subjects
aged 41 to 50 years, and 28% in the 17 subjects older than 50 years. In hemophiliacs, there was a clear cut increase of TTV-DNA prevalence in the group older than 10 years (76% v 32%, P < .001). However, when the 28 patients younger than 10 years were
stratified by treatment modalities, serum TTV-DNA was detected only in
2 of 16 (12%) who were exclusively treated by recombinant factors
compared with 7 of 12 (58%) of those who had received recombinant and
virus-inactivated factors (P = .01).
| |
DISCUSSION |
The TT virus was initially identified as a transfusion-transmissible
agent present in a large number of patients with acute and chronic
hepatitis of non-A to G etiology.9,10 This virus was also
reported in severe hemophiliacs in Scotland, Japan, and France who were
recipients of TTV-infected batches of clotting factor concentrates
manufactured from large plasma pools.14-16 Sixty-nine
percent of the hemophilic patients evaluated in this study circulated
TTV-DNA compared with 22% healthy blood donors. Unlike HBV, HCV, and
HIV infections that prevailed in the recipients of unmodified
concentrates, TTV was equally common in the recipients of unmodified
concentrates and in those treated with virus-inactivated factors only
(78% and 67%, respectively). The prevalence of TTV viremia in our
Italian hemophiliacs, even in the recipients of virus-inactivated
clotting concentrates, was higher than that reported for patients from
other countries. A study in France16 reported a 30%
prevalence of TTV-DNA among recipients of virus-inactivated concentrates, whereas the studies in UK and Japan demonstrated lower
rates of serum TTV-DNA among recipients of virus-inactivated products
than in recipients of unmodified concentrates (5% and 43% v
27% and 78%, respectively).14,15
TTV is a nonlipid-enveloped virus and, therefore, it might be present
in pooled coagulation factor-concentrates, virally-inactivated with
solvent/detergent method. In vitro studies have shown that solvent/detergent is less effective than heating (pasteurization at
60°C for 10 hours) in inactivating TTV.14 As many as
two thirds of our patients have been treated with
solvent/detergent-inactivated products. Yet, the higher rate of TTV
viremia observed in our patients is unlikely to be due exclusively to
differences in efficiency of virus-inactivation procedures, because
comparable prevalence of TTV was observed in patients who received
clotting factors treated with other procedures.15,16 Both
blood product-related and nonparenteral, community-acquired infections
could contribute to the high frequency of TTV in the Italian
hemophiliacs, consistent with a higher prevalence of TTV in Italian
blood donors compared with those in other western countries. A
potential fecal transmission route of TTV has been
suggested.22 In this study, TTV-DNA was detected in 22%
healthy blood donors, despite the fact that this population was
selected on the basis of being at low risk of blood-borne infections.
High prevalence (>90%) of TTV-DNA also has been observed in
multitransfused Italian thalassemic patients,13 however, the precise reasons for a higher carrier rate in Italy is presently unclear.
In our patients, the prevalence of HCV-RNA and anti-HIV was
significantly greater in patients treated with unmodified concentrates (85%) than in recipients of virus-inactivated (15%) or recombinant concentrates (0%). Anti-HIV and HBsAg were only detected in recipients of unmodified concentrates, 24% and 5%, respectively. Hepatitis G
viral RNA, however, was rarely detected (0% to 8%) in hemophiliacs, regardless of the source of clotting factors. Interestingly, the risk
of acquiring TTV alone was greater for the recipients of unmodified
concentrates than for the recipients of recombinant factors when
compared with patients who received virus-inactivated concentrates
(relative risk [RR], 1.24; 95% CI, 0.27 to 5.79 v 0.09; 95% CI, 0.01 to 0.52). The corresponding figures for
the risk of acquiring any HCV, HBV, HGV, or HIV were 41.4 (95% CI, 9.3 to 204.5) and 0 (95% CI, 0 to 0.82), respectively.
In previous studies, the potential of TTV causing hepatitis was
implicated by the association of TT viremia with ALT
elevation.9,17 In this study, biochemical signs of liver
disease were present in half the patients, but only in 7% of those
exclusively infected by TTV. The possibility of underestimating
TTV-related liver damage due to the imperfect diagnostic accuracy of
serum ALT patterns, in patients with transfusion-related hepatitis,
cannot be ruled out, as we learned from HCV-infected
patients.23 Taken together, however, results in this study
do not suggest a causal effect of TTV on chronic liver disease of
unknown etiology in this small population (7%) of TTV-infected
hemophiliacs. There is also no evidence of the virus affecting the
degree of liver damage when present as coinfection with HBV or HCV.
Nevertheless, association of TTV with transient elevation of ALT and
its potential for causing hepatitis has been reported in transfusion
recipients and immunocompromised organ-transplant
patients.17 A better understanding of the pathogenicity of
TTV and the use of recombinant clotting factor should reduce any
further hepatic complications in hemophilic patients.
| |
ACKNOWLEDGMENT |
The authors thank Dr R. Bohenzky and the sequencing-informatics team at
Sentinel Biosciences, Inc for their expert technical support; Professor
J.P. Allain and Dr H. Lee for their helpful discussion and
encouragement, and Dr A. Russo for statistical analysis.
| |
FOOTNOTES |
Submitted April 15, 1999; accepted August 9, 1999.
Supported by a grant from Istituto Superiore di Sanità
(96/B/T24).
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
1734 solely to indicate this fact.
Address reprint requests to Massimo Colombo, MD, Professor and
Chairman, Department of Internal Medicine, Via Pace 9, 20122 Milan,
Italy; e-mail: mcolombo{at}imiucca.csi.unimi.it.
| |
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ABSTRACT
INTRODUCTION