TT virus infection in haemodialysis patients

Nadia Campo1, Renata Brizzolara1, Nicoletta Sinelli1, Francesco Torre1, Rodolfo Russo2, Giacomo Deferrari2 and Antonino Picciotto1,

Department of Internal Medicine, 1 Gastroenterology Unit and 2 Nephrology Unit, University of Genoa, Genoa, Italy



   Abstract
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 Abstract
 Introduction
 Patients and methods
 Methods
 Results
 Discussion
 References
 
Background. The recent discovery of a new parenterally transmitted DNA virus called TT virus (TTV) led us to investigate its prevalence in haemodialysis patients, a high-risk group for blood-borne infection, and to evaluate its role in liver disease. Moreover, we compared the TTV prevalence with the prevalence of other hepatitis virus coinfections.

Methods. Serum samples of 78 patients on maintenance haemodialysis were tested for TTV-DNA, hepatitis G virus (HGV)-RNA, anti-E2, anti-hepatitis C virus (HCV) and HCV-RNA. TTV-DNA was detected by semi-nested PCR using the primers from open reading frame 1 (ORF). HGV-RNA was detected by PCR using specific primers for the NS3 and the 5'-UTR genome regions while anti-E2 were checked by an enzyme immunological test. Anti-HCV was tested by the second generation Chiron RIBA HCV test system. HCV-RNA was evaluated by nested PCR with primers directed to the highly conserved 5' non-coding region of the HCV genome.

Results. TTV prevalence in our patients was 19% (15/78) while the prevalence of HCV and HGV infection proved to be 20 and 15.4%, respectively. Among TTV positive patients HGV co-infection was present in five cases (33%), HCV in six cases (39.9%), while HBV co-infection was not present in any of the patients. Only three patients proved positive for all three viruses. ALT levels were normal in most cases (13/15; 86%). In particular, patients with TTV infection alone showed normal ALT levels and HCV coinfection was found in the two patients with moderate ALT increases.

Conclusions. TTV prevalence in haemodialysed patients is significant though the real clinical impact is still unclear. However, we must keep in mind that the epidemiological relevance of TTV infection is probably underestimated due to the impossibility in detecting the corresponding antibody.

Keywords: TTV-DNA; TTV; HGV-RNA; HGV antibody; hepatitis C virus; haemodialysis



   Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Methods
 Results
 Discussion
 References
 
Patients on maintenance haemodialysis are at high risk for liver infection as a result of blood-borne viral agents. At present hepatitis C virus (HCV) is the most common cause of viral hepatitis in these patients [1,2]. Epidemiological data showed a considerable seroprevalence of hepatitis G virus (HGV) among dialysed patients when HGV viraemia and anti-E2 were tested [35]. However, evidence of liver disease due to this virus was not confirmed in the majority of these studies [6]. Recently, a novel DNA virus called TT virus (TTV) has been reported in association with post-transfusion and acute hepatitis of unknown aetiology [79]. Available data showed the presence of TTV among polytransfused patients with liver disease of unknown aetiology with a prevalence ranging from 25 to 46% [8,10]. Moreover, TTV was reported as inducing fulminant hepatitis [10,11].

The few data available concerning TTV infection among haemodialysis patients show a high prevalence 32% [12].

The aim of our study was to detect TTV-DNA in a group of haemodialysis patients and to evaluate its clinical impact, taking into account co-infection with other hepatitis viruses.



   Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Methods
 Results
 Discussion
 References
 
Patients
We studied 78 patients (males 45/females 33; age 62±14 years) on maintenance haemodialysis at the Nephrology Unit of our Department. The duration of haemodialysis (mean±SD) was 52±51 months. Three patients in our series were HBsAg positive and 24 patients (30.7%) had received multiple transfusions. Sera were tested for anti-HCV, HCV-RNA, anti-E2, HGV-RNA and for TTV-DNA. Alanine aminotransferase (ALT) levels were assessed routinely during haemodialysis. Informed consent was obtained from each patient.



   Methods
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 Abstract
 Introduction
 Patients and methods
 Methods
 Results
 Discussion
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Serological assay
Anti-HCV was tested by the second generation Chiron RIBA HCV test system. HBsAg, anti-HBs and anti-HBc were determined by commercial radioimmunoassay (RIA) kit (Abbott Laboratories, North Chicago, IL, USA).

Viral-RNA extraction
Sera were stored at -80°C within 2 h of withdrawal. Nucleic acids were extracted from 140 µl of serum according to Kit QIAamp HCV (QIAGEN) assay and subsequently resuspended in RNAase-free water. Total RNA was primed with random hexamers and reverse transcription to cDNA was performed at 42°C for 30 min (AMV, Boehringer Mannheim).

HCV-RNA detection
cDNA was amplified by using a nested PCR method with four primers complementary to the highly conserved 5' non-coding region of the HCV genome [13].

HGV-RNA detection
cDNA was subjected to PCR using specific primers (0.2 µM final concentration) for the NS3 and the 5'-UTR genome regions in all serum samples.

5'-UTR region.
The first round of PCR was performed using G1, 5'-AAGCCCCAGAAACCGACGCC-3', as sense primer and G2, 5'-TGAAGGGCGACGTGGACCGT-3', as antisense primer. Nested PCR was performed using G3, 5'-CGGCCAAAAGGTGGTGGATG-3', as sense primer and G4, 5'-GTAACGGGCTCGGTTTAACG-3', as antisense primer [14]. Cycling conditions were: 40 cycles for the first round (consisting of denaturation for 60 s at 94°C, annealing for 60 s at 55°C and extension for 90 s at 72°C) followed by an extension at 72°C for 5 min; 35 cycles for the second round, under the same conditions (DNA Thermal Cycler 480, Perkin-Elmer Cetus).

NS3/helicase region.
The first PCR was performed using G8, 5'-TATGGGCATGGHATHCCYCT-3' (H=A, C or T; Y=C or T) as sense primer and G9, 5'-TCYTTGATGATDGAACTGTC-3' (Y=T or C; D=A, G or T) as antisense primer. Nested PCR was performed using a semi-nested protocol with G8 as sense primer and G11, 5'-TCYTTACCCCTRTAATAGGC-3' (Y=C or T; R=A or G) as antisense primer [14]. Forty cycles were run for the first round (consisting of denaturation for 60 s at 94°C, annealing for 60 s at 57°C and extension for 90 s at 72°C) followed by an extension at 72°C for 5 min; 35 cycles were performed for the semi-nested PCR, under the same conditions (DNA Thermal Cycler 480, Perkin-Elmer Cetus).

PCR products were subjected to electrophoresis on 2% agarose gel (Agarose LE, Genenco). The expected size for the PCR products to be derived from NS3 and 5'-UTR were 140 and 204 bp, respectively with reference to the standard molecular weight (174 RF DNA/HaeIII).

Sequence analysis (Amplicycle Sequencing Kit, Perkin-Elmer) was performed using G4 and G11 as sequencing primers for both genomic areas in the two PCR positive haemodialysis patients to confirm the specificity of the PCR amplified products. The nucleotide sequences of the 5'-UTR region showed 100% homology with the HGV sequence (GeneBank accession number U44402). The sequence of the NS3 region showed 95% homology.

Detection of anti-E2
The serum qualitative determination of IgG antibodies to the HGV E2-antigen was performed by the enzyme immunological test (µPLATE Anti Hgenv, Boehringer Mannheim).

Detection of TTV
DNA was extracted from 200 µl serum using QIAmp Blood kit (QIAGEN Ltd, Crawley, UK) and resuspended in 50 µl elution buffer. TTV-DNA was amplified by semi-nested PCR with TTV-specific primers derived from two conserved regions according to the published sequences [8].

The first round of PCR was performed using primer TTV1 (sense primer 5'-ACAGACAGAGGAGAAGGCAAYATG -3') and primer TTV3 (antisense primer 5'-CTGGCATTTTACCATTTCCAAAGTT-3'). Amplification was for 35 cycles at 94°C for 45 s, 58°C for 30 s and 72°C for 45 s, followed by 4 min at 72°C. The second round was performed with another sense primer TTV2 (5'-GGCAACATGTTATGGATAGACTGG-3') and the same antisense primer TTV3 using 1 µl of the first round PCR. Amplification was obtained by the same method. The amplicons were electrophoresed in 1% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. Sequence by Sanger method using inner primer was used to confirm the specificity of the amplification products.



   Results
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 Abstract
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 Patients and methods
 Methods
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In our series (Table 1Go), TTV infection was present in 19.2% of the patients (15/78), while the prevalence for HCV and HGV infection was 33.3 (26/78) and 15.4% (12/78), respectively. HCV-RNA was detected in 15 out of 26 anti-HCV positive patients. Among HGV infected patients, only two were HGV-RNA positive (16.6%). In 37 out of 78 patients on haemodialysis no blood-borne viral infections have been detected (Table 2Go).


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Table 1. Characteristics of TTV-DNA ±ve and TTV-DNA -ve patients

 

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Table 2. Characteristics of patients without viral infections

 
Within the group of TTV positive cases, seven patients out of 15 had this infection alone (46.6%). Co-infection with HCV was present in six patients (40 versus 31.7% in the 20 TTV negative cases) three of whom were HCV-RNA positive. HGV co-infection was detected in six cases (40 versus 9.5% in the six TTV negative-group cases), five were anti-E2 positive and only one was HGV-RNA positive. The simultaneous presence of TTV, HCV and HGV was detected in four cases (26.6%). None of the TTV +ve patients was HBsAg carrier.

Patients with TTV infection alone showed normal ALT levels while HCV co-infection was present in the two patients with moderate ALT elevation.

We found a high prevalence of history of transfusion among TTV-DNA +ve patients (8/15; 53.3%) when compared with TTV-DNA -ve patients (16/63; 25.3%; P<0.05; {chi}2 test). No statistically significant differences were present between these groups for what concern age, sex, time on haemodialysis, liver function test or prevalence of HCV or HGV infection (Table 1Go).



   Discussion
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 Abstract
 Introduction
 Patients and methods
 Methods
 Results
 Discussion
 References
 
A new DNA virus named TTV was recently identified in Japan in the serum of patients with post-transfusion non-A–G hepatitis. The discovery of another virus potentially causing liver disease induced us to study the TTV seroprevalence in patients at high risk for parenterally transmitted agents. Currently few data are available regarding TTV infection among haemodialysed patients. Our data show that the TTV is common among our haemodialysis patients with a prevalence of 19%, although this percentage is lower than what was reported in previous Japanese study [12].

A possible explanation for this difference may be the different spreading of infection between the two geographic areas. Moreover, taking into account the existence of different previously described TTV genotypes the geographic, genotypic prevalence may underestimate the real diffusion of infection in our area [1518].

The percentage of patients who have undergone multiple transfusions was higher in the TTV positive group (eight out of 15 patients, 53.3%) than in the TTV negative group (16 out of 63 patients, 25.3%) suggesting that the parenteral route may play an important role in transmitting the virus. In fact when haemodialysed patients were grouped according to their history of multiple transfusions, TTV infection was present in eight of the 24 polytransfused patients (33.3%) and in seven of the 54 non-transfused ones (12.9%). Moreover, the TTV infection alone was present in four polytransfused patients (16.6%), but only in three (5.5%) of the non-transfused patients. We did not find any difference related to age, time on haemodialysis, liver function test or prevalence of HCV or HGV infection between TTV +ve or -ve patients, as previously reported [1219,20].

TTV infection in the non-transfused patients might be related to dialysis or to a non-parenteral way as has previously been described. In fact, the discovery of TTV excretion in the faeces suggests that the infection may be transmitted by faecal–oral route [21]. Regarding possible sexual transmission, a low percentage of TTV infection in groups of subjects at high risk for this kind of transmission was observed [22]. Moreover, the importance of non parenteral transmission of TTV is supported by the higher percentage of infection than other hepatitis viruses among blood donors [23,24].

As far as the clinical impact of TTV infection is concerned, our data show that the patients infected with TTV alone had normal ALT levels while the biochemical alterations in the TTV positive cases with moderate ALT increase may be accounted for by HCV co-infection, as already observed [25,26]. Presently the lack of tests to detect antibodies against TTV, may cause an underestimation of the real diffusion of TTV infection and, thus, its epidemiological impact. No data are available regarding the prevalence of TTV infection in a control population in our area. However, in another group of patients at risk of parenteral transmission (anti-HCV positive, but without history of transfusion), we observed a low prevalence of TTV infection (four out 65 patients, 6.2% unpublished data). Furthermore, recent data from an Italian survey on blood donors reports a prevalence of TTV infection of 5±1.9% in our country [27].

In conclusion, although TTV prevalence in haemodialysed patients is significant, the real clinical impact is still unclear. In fact our data did not show that when TTV alone is present it induces liver function tests alteration. However, it must be taken into account that the epidemiological relevance of TTV infection is probably underestimated due to the difficulty in detecting the corresponding antibody.



   Notes
 
Correspondence and offprint requests to: Prof. A. Picciotto, Department of Internal Medicine, Gastroenterology Unit, University of Genoa, Viale Benedetto XV, 6, I-16132 Genoa, Italy. Back



   References
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 Abstract
 Introduction
 Patients and methods
 Methods
 Results
 Discussion
 References
 

  1. Picciotto A, Varagona G, Gurreri G et al. Anti-hepatitis C virus antibodies and hepatitis C virus viraemia in haemodialysis patients. Nephrol Dial Transplant1993; 8: 1115–1117[Abstract]
  2. Pereira BJ, Levey AS. Hepatitis C virus infection in dialysis and renal transplantation. Kidney Int1997; 51: 981–999[ISI][Medline]
  3. Masuko K, Mitsui T, Iwano K et al. Infection with hepatitis GB virus C in patients on maintenance hemodialysis. N Engl J Med1996; 334: 1485–1490[Abstract/Free Full Text]
  4. De Lamballerie X, Charrel RN, Dussol B. Hepatitis GB virus C in patients on hemodialysis. N Engl J Med1996; 334: 1549[Free Full Text]
  5. Campo N, Sinelli N, Brizzolara R et al. Hepatitis G virus infection in haemodialysis and peritoneal dialysis patients. Nephron1999; 82: 17–21[ISI][Medline]
  6. Pessoa MG, Terrault NA, Detmer J et al. Quantitation of hepatitis G and C viruses in the liver: evidence that hepatitis G virus is not hepatotropic. Hepatology1998; 27: 877–880[ISI][Medline]
  7. Nishizawa T, Okamoto H, Konishi K. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem Biophys Res Commun1997; 241: 92–97[ISI][Medline]
  8. Okamoto H, Nishizawa T, Kato N et al. Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown etiology. Hepatol Res1998; 10: 1–16[ISI]
  9. Simmonds P, Davidson F, Lycett C et al. Detection of a novel DNA virus (TTV) in blood donors and blood products. Lancet1998; 352: 191–194[ISI][Medline]
  10. Naoumov N.V, Petrova E.P, Thomas M.G, Williams R. Presence of a newly described human DNA virus (TTV) in patients with liver disease. Lancet1998; 352: 195–197[ISI][Medline]
  11. Charlton M, Adjei P, Poterucha J et al. TT-virus infection in North American blood donors, patients with fulminant hepatic failure and cryptogenic cirrhosis. Hepatology1998; 28: 839–884[ISI][Medline]
  12. Oguchi T, Tanaka E, Orii K, Kobayashi M, Hora K, Kiyosawa K. Transmission of and liver injury by TT virus in patients on maintenance hemodialysis. J Gastroenterol1999; 34: 234–240[ISI][Medline]
  13. Han JH, Shjamala V, Richman KH et al. Characterization of the terminal regions of Hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly (A) tails at the 3' end. Proc Nat Acad Sci USA1991; 88: 1711–1715[Abstract]
  14. Schreirer E, Hohne M, Kunkel U, Berg T, Hopf U. Hepatitis GBV-C sequences in patients infected with HCV contaminated anti-D immunoglobulin and among i.v. drug users in Germany. IX Triennal International Symposium on viral hepatitis and liver disease, April 21–25, 1996; Rome, Italy; 258
  15. Viazov S, Ross RS, Niel C et al. Sequence variability in the putative coding region of TT virus: evidence for two rather than several major types. J Gen Virol1998; 79: 3085–3089[Abstract]
  16. Okamoto H, Kato N, Iizuka H, Tsuda F, Miyakawa Y, Mayumi M. Distinct genotypes of a nonenveloped DNA virus associated with posttransfusion non A to G hepatitis (TT virus) in plasma and peripheral blood mononuclear cells. J Med Virol1999; 57: 252–258[ISI][Medline]
  17. Takayama S, Yamazaki S, Matsuo S, Sugii S. Multiple infection of TT virus (TTV) with different genotypes in Japanese hemophiliacs. Biochem Biophys Res Commun1999; 56: 208–211
  18. Tanaka Y, Mizokami M, Orito E et al. A new genotype of TT virus (TTV) infection among Colombian native Indians. J Med Virol1999; 57: 264–268[ISI][Medline]
  19. Gallian P, Berland Y, Olmer M et al. TT virus infection in French hemodialysis patients: study of prevalence and risk factors. J Clin Microbiol1999; 37: 2538–2542[Abstract/Free Full Text]
  20. Maggi F, Fornai C, Morrica A et al. High prevalence of TT virus viremia in Italian patients, regardless age, clinical diagnosis, and previous interferon treatment. J Infect Dis1999; 180: 838–842[ISI][Medline]
  21. Okamoto H, Akahane Y, Ukita M et al. Fecal excretion of a nonenveloped DNA virus (TTV) associated with posttransfusion non A–G hepatitis. J Med Virol1998; 56: 128–132[ISI][Medline]
  22. MacDonald DM, Scott GR, Clutterbuck D, Simmonds P. Infrequent detection of TT virus infection in intravenous drug users, prostitutes, and homosexual men. J Infect Dis1999; 179: 686–689[ISI][Medline]
  23. Tanaka H, Okamoto H, Luengrojanakul P, Chainuvati T, Tsuda F, Tanaka. Infection with an unenveloped DNA virus (TTV) associated with posttransfusion non-A to G hepatitis in hepatitis patients and healthy blood donors in Thailand. J Med Virol1998; 56: 234–238[ISI][Medline]
  24. Neil C, de Oliveira JM, Ross RS, Gomes SA, Roggendorf M, Viazov S. High prevalence of TT virus infection in Brazilian blood donors. J Med Virol1999; 57: 259–263[ISI][Medline]
  25. Yuki N, Kato M, Masuzawa M et al. Clinical implications of coinfection with a novel DNA virus (TTV) hepatitis C virus carriers on maintenance hemodialysis. J Med Virol1999; 59: 431–436[ISI][Medline]
  26. Forns X, Hegerich P, Darnell A, Emerson SU, Purcell RH, Bukh J. High prevalence of TT virus (TTV) infection in patients on maintenance hemodialysis: frequent mixed infections with different genotypes and lack of evidence of associated liver disease. J Med Virol1999; 59: 313–317[ISI][Medline]
  27. Pisani G, Antigoni I, Bisso G et al. Prevalence of TT viral DNA in Italian blood donors with and without elevated serum ALT levels: molecular characterization of viral DNA isolates. Haematologica2000; 85: 181–185[ISI][Medline]
Received for publication: 12. 7.99
Revision received 21. 6.00.