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BRIEF REPORT
From the Public Health Department, Microbiology and
Virology Unit, University of Florence, Italy; the Hematology Department
and Hemophilia Center, Azienda Ospedaliera Careggi, Florence, Italy;
the Hematology Department and Hemophilia Center of Catania, Italy; the
A. Bianchi Bonomi Hemophilia Center, IRCCS Maggiore Hospital and
University of Milan, Italy; and the Hematology Department and
Hemophilia Center, San Bortolo Hospital, Vicenza, Italy.
Recombinant factor VIII and factor IX concentrates,
human-plasma-derived albumin, and samples from previously untreated
patients with hemophilia were examined for the presence of TT virus
(TTV) by using polymerase chain reaction testing. Blood samples from the patients were obtained prospectively before and every 3 to 6 months
after therapy was begun. TTV was detected in 23.5% of the
recombinant-product lots and 55.5% of the albumin lots tested. Only
first-generation factor VIII recombinant concentrates stabilized with
human albumin were positive for TTV, whereas all second-generation (human protein-free) concentrates were negative for the virus. In 59%
of patients treated with either first- or second-generation recombinant
factor concentrates, TTV infection developed at some point after the
initial infusion. Infection with TTV in these patients before and after
treatment did not appear to be clinically important. Thus,
first-generation recombinant factor VIII concentrates may contain TTV
and the source of the viral contamination may be human albumin.
(Blood. 2001;98:2571-2573) TT virus (TTV) is a small, naked, single-stranded
DNA virus of the Circoviridae family with a circular genome. It was
first detected in the serum of patients with posttransfusion hepatitis of unknown cause.1 It is not known whether TTV is
pathogenic.2-4 TTV may be transmitted parenterally through
transfusion of blood products,5-7 by the fecal-oral route,
and by pregnant women to fetuses.8,9 TTV may also be
disseminated by aerosols, since the virus is present in
saliva.8,10 A high rate of TTV infection was found in
patients given multiple transfusions11 and patients with
hemophilia.5,12-14 TTV sequences are frequently detected in clotting factor concentrates prepared from large plasma
pools.5,15
First-generation recombinant human factor VIII concentrates stabilized
with human-plasma-derived albumin (HPDA) before lyophilization (Recombinate, Baxter, Glendale, CA; and Kogenate, Bayer, Leverkusen, Germany) have been licensed for more than 10 years. They became widely
used by patients with hemophilia because of their perceived safety with
respect to viral infection. However, parvovirus B19 DNA was detected by
polymerase chain reaction (PCR) assay in a few lots of
Kogenate,16 and seroconversion occurred in a few patients
with hemophilia who were treated with this product.17 The
source of the B19 virus contamination in recombinant products was
thought to be the HPDA used to stabilize the recombinant protein before
lyophilization. Second-generation recombinant factor VIII and factor IX
concentrates that do not contain albumin (ReFacto and Benefix, Genetics
Institute, Cambridge, MA) were recently licensed for the treatment of
hemophilia. A prospective study of the viral safety of these
concentrates in accordance with International Society on Thrombosis and
Haemostasis guidelines has not yet been conducted. To assess the safety
of recombinant concentrates regarding TTV transmission, we tested
several such products for TTV DNA by using PCR assays. We also studied
the incidence of TTV infection in 24 previously untreated patients
before and after treatment with recombinant clotting factor concentrates.
Recombinant clotting factor concentrates and human albumin
Patients
PCR for detection of TTV DNA Before amplification, the samples (recombinant products, HPDA, and serum) were subjected to a procedure for DNA extraction (Roche, Basel, Switzerland) to eliminate PCR inhibitors. Three different nested PCRs (untranslated region [UTR] (A) PCR, UTR (B) PCR, and N22-PCR), which amplify distinct viral DNA regions, were used to detect TTV DNA. The first and second reactions amplify 2 untranslated, more conserved, regions, whereas the third target sequence is in the N22 region,18 is more variable, and is in the coding region of the TTV genome. The UTR (A) PCR was done with NG133 and NG147 as outer primers and NG134 and NG132 as inner primers. For the UTR (B) PCR, the outer primers were NG148 and NG135 and the inner primers were NG149 and NG136; and for the N22 PCR, the outer primers were NG059 and NG063 and the inner primers were NG061 and NG063. The PCR conditions were described previously.18 Samples positive on the UTR (A) PCR assay only were considered positive after repeated control assessments.
TTV DNA was present in 8 of the 34 recombinant-product lots (Table
1). Specifically, TTV DNA was found in
35% of recombinant-product lots to which albumin had been added as a
stabilizer before lyophilization (Recombinate and Kogenate). The
albumin-free products (Benefix and ReFacto) were negative for TTV DNA.
Thus, it appeared that albumin was the source of the TTV contamination.
Five of the 9 lots of albumin tested (55.5%) were positive for
TTV DNA.
Of the 8 TTV-positive recombinant products, 2 were positive on the UTR (A) PCR assay only, 5 on both the UTR (A) and UTR (B) PCR assay, and 1 on all 3 PCR tests. Four lots of HPDA were positive on both the UTR (A) and UTR (B) PCR assay, and 1 was positive on the UTR (A) PCR assay only. This result only apparently contradicts findings by Pisani et al,15 who did not detect TTV DNA sequences in HPDA by using primers for the N22 region. The low percentage of positive results on the N22 PCR assay was likely due to the high variability of the viral genome in this region and not to a different sensitivity of the 3 PCR reactions, in agreement with previously reported data.18 The study of previously untreated patients (Figure
1) showed that baseline samples from 7 of
the 24 patients (29%) were positive for TTV DNA, probably because of
early spread of TTV infection through fecal-oral or maternal-fetal
transmission. TTV DNA was detected in 7 of 17 patients negative for TTV
(41%) in the first serum sample obtained 3 to 6 months after the first
infusion of a recombinant product. Serum samples from 11 of 17 TTV-negative patients obtained after subsequent infusions were
available for analysis. In 3 patients with hemophilia who were negative
for TTV after the first treatment, TTV viremia developed after
subsequent infusions. Overall, serum samples showing TTV viremia were
obtained after the first or one of the subsequent infusions in 10 patients (59%)
For several reasons, we could not demonstrate that TTV was transmitted by the recombinant products. Moreover, the serum samples showing TTV viremia were obtained from patients after treatment with either first-generation albumin-containing products or second-generation, albumin-free recombinant products. All patients had normal results on clinical chemistry tests at enrollment, and no patient had alterations in levels of transaminases, GGT, or bilirubin. No clinical signs of acute illness were observed during the follow-up period. TTV may have little or no pathogenicity. However, detection of TTV DNA in recombinant products containing HPDA as a stabilizer, regardless of its clinical relevance, indicates a need for additional procedures to ensure product safety. Furthermore, viral surveillance in noninfected patients with hemophilia must be conducted regardless of whether they are being treated with recombinant clotting factor concentrates, since assuming that these products are completely free of virus may be misleading.
We thank Nadia Ewing, MD, Division of Pediatrics, City of Hope National Medical Center, Duarte, CA, for critically reviewing the manuscript.
Submitted February 8, 2001; accepted June 8, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Alberta Azzi, Dipartimento di Sanità Pubblica (Sezione di Microbiologia e Virologia), Viale Morgagni 48, 50134 Florence, Italy; e-mail: azzi{at}dsp.igiene.unifi.it.
1. Nishizawa T, Okamoto H, Konishi K, Yoshizawa H, Miyakawa Y, Mayumi M. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem Biophys Res Commun. 1997;241:92-97[CrossRef][Medline] [Order article via Infotrieve].
2.
Bendinelli M, Pistello M, Maggi F, Fornai C, Freer G, Vatteroni ML.
Molecular properties, biology, and clinical implication of TT virus, a recently identified widespread infectious agent of humans.
Clin Microbiol Rev.
2001;14:98-113 3. Gimenez-Barcons M, Forns X, Ampurdanes S, et al. Infection with a novel human DNA virus (TTV) has no pathogenic significance in patients with liver diseases. J Hepatol. 1999;30:1028-1034[CrossRef][Medline] [Order article via Infotrieve]. 4. Huang Y, Wu J, Chiang T, et al. Detection and viral nucleotide sequence analysis of transfusion-transmitted virus infection in acute fulminant and non-fulminant hepatitis. J Viral Hepatol. 2000;7:56-63[Medline] [Order article via Infotrieve]. 5. Simmonds P, Davidson F, Lycett C, et al. Detection of a novel DNA virus (TTV) in blood donors and blood products. Lancet. 1998;352:191-195[CrossRef][Medline] [Order article via Infotrieve].
6.
Lefrere JJ, Roudot-Thoraval F, Lefrere F, et al.
Natural history of the TT virus infection through follow-up of TTV DNA-positive multiple-transfused patients.
Blood.
2000;95:347-351 7. Allain JP. Emerging viral infections relevant to transfusion medicine. Blood Rev. 2000;14:173-181[CrossRef][Medline] [Order article via Infotrieve]. 8. Ross SS, Viazov S, Runde V, Schaefer UW, Roggendorf M. Detection of TT virus DNA in specimens other than blood. J Clin Virol. 1999;13:181-184[CrossRef][Medline] [Order article via Infotrieve].
9.
Schroter M, Polywka S, Zollner B, Schafer P, Laufs R, Feucht HH.
Detection of TT virus DNA and GB virus type C/hepatitis G virus RNA in serum and breast milk: determination of mother-to-child transmission.
J Clin Microbiol.
2000;38:745-747
10.
Inami T, Konomi N, Arakawa Y, Abe K.
High prevalence of TT virus DNA in human saliva and semen.
J Clin Microbiol.
2000;38:2407-2408 11. Maeda M, Hamada H, Tsuda A, Kaneko K, Fukunuga Y. High rate of TTV infection in multitransfused patients with pediatric malignancy and hematological disorders. Am J Hematol. 2000;65:41-44[CrossRef][Medline] [Order article via Infotrieve].
12.
Chen BP, Rumi MG, Colombo M, et al.
TT virus is present in high frequency of Italian hemophilic patients transfused with plasma-derived clotting factor concentrates.
Blood.
1999;94:4333-4336 13. Maggi F, Fornai C, Morrica A, et al. High prevalence of TT virus viremia in Italian patients, regardless of age, clinical diagnosis and previous interferon treatment. J Infect Dis. 1999;180:838-842[CrossRef][Medline] [Order article via Infotrieve]. 14. Yokozaki S, Toyoda H, Nakano I, et al. Infection with TT virus, a novel transfusion-transmissible DNA virus, in haemophiliacs and in blood products. Br J Haematol. 1999;105:1114-1119[CrossRef][Medline] [Order article via Infotrieve]. 15. Pisani G, Cristiano K, Wirz G, et al. Prevalence of TT virus in plasma pools and blood products. Br J Haematol. 1999;106:431-435[CrossRef][Medline] [Order article via Infotrieve]. 16. Eis-Hubinger AM, Sasowski U, Brackmann HH, et al. Parvovirus B19 is frequently present in recombinant coagulation factor VIII products. Thromb Haemost. 1996;76:1120[Medline] [Order article via Infotrieve]. 17. Aygoren-Pürsün E, Scharrer I. A multicenter pharmacosurveillance study for the evaluation of the efficacy and safety of recombinant factor VIII in the treatment of patients with hemophilia A. German Kogenate Study Group. Thromb Haemost. 1997;78:1352-1356[Medline] [Order article via Infotrieve]. 18. Okamoto H, Takahashi M, Nishizawa T, et al. Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and noncoding regions. Virology. 1999;259:428-436[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  T. R. Kreil, K. Zimmermann, S. Pable, H. P. Schwarz, F. Dorner, A. Azzi, and M. Morfini TT virus does not contaminate first-generation recombinant factor VIII concentrate Blood, August 28, 2002; 100(6): 2271 - 2271. [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
20°C until the PCR assay.
-glutamyltransferase [GGT], and bilirubin) and for the TTV PCR
assays were collected before and after the first infusion and at
intervals ranging from 3 to 6 months. The total follow-up time was at
least 3 years.
6 patients treated with first-generation recombinant
products and 4 patients treated with second-generation products. At the end of the follow-up period, only 6 patients were positive for TTV DNA;
the other 4 had clearance of the virus. Of the 7 patients who were
positive for TTV DNA at baseline, 5 were positive at the end of the
follow-up period and 2 were negative. TTV viremia persisted
intermittently or continually for 1 to 3 years in 7 patients.
