TT Virus infection in the elderly

Transcription

1 JOURNAL OF PREVENTIVE MEDICINE AND HYGIENE 1999; 40: ORIGINAL ARTICLE TT Virus infection in the elderly A. FLOREANI, V. BALDO 1, S. BUORO 2, L. MAZZARIOL 3, M. FAVARATO 3, R. TRIVELLO 2 AND COLLABORATIVE GROUP: W. Boato 3, M. Carletti 4, M. Cristofoletti 4, M. Miorin 4, S. Perale 3, L. Rossi 3, C. Scalici 3, A. Vesco 3 Department of Surgical and Gastroenterological Sciences, University of Padua, Italy 1 Department of Environmental Medicine and Public Health, Institute of Hygiene, University of Padua, Italy 2 Hospital of Dolo, Section of molecular biology, Local Health Unit n. 13, Veneto Region, Italy 3 «Mariutto» Institute for the Elderly, Mirano (Venice), Italy 4 Epidemiology Service, Local Health Unit 13, Veneto Region, Italy Key words TTV Elderly HBV HCV Epidemiology An epidemiological survey was carried out in an old people s nursing home. The aims of the present study were therefore: i) to evaluate the prevalence of TTV infection in a sample of institutionalized elderly subjects; ii) to correlate TTV prevalence with HBV and HCV seroprevalence; iii) to correlate TTV prevalence with any underlying diseases and conditions. The overall sample included 285 subjects with a mean age of 83.4 ± 9.1 years (range years). Twenty-six subjects (9.5%) were found TTV-DNA positive. The highest rate of TTV positivity was found in the Summary year-old age bracket (10.9%), though no correlation was found with advancing age (p = 0.764) or with the length of institutionalization (p = 0.217). Coinfection was found with HBsAg in one subject and with HCV in two. The rate of HCV infection was 11.6%. Multivariate analysis showed that diabetes (p = 0.03) and self-sufficiency (p < 0.001) were independent factors associated with TTV. None of the subjects had altered liver function tests or evidence of liver disease. TTV infection is unassociated with any evidence of liver damage, and its pathogenic role is questionable. Introduction A new DNA virus was isolated in a serum sample from a Japanese patient with post-transfusion hepatitis of unknown etiology 1. This agent was named TTvirus (TTV) from the initials of the patient concerned. The TTV genome is a single-stranded DNA of about 3739 nucleotides and contains two overlapping open reading frames (ORF1 and ORF2) 2 3. TTV can be transmitted parenterally by blood products 3-5 and probably by a fecal-oral route 5 6. TTV DNA has been detected in patients with fulminant hepatitis, in acute hepatitis of various etiologies, and also in chronic liver diseases. Its prevalence in such liver diseases is variable and often conflicting. Moreover, TTV DNA has been detected in a considerable proportion (1.9%-12%) of blood donors in several countries To our knowledge, there are no reports in the literature dealing with TTV infection in the elderly though, from the epidemiological point of view, these subjects represent the age group with the highest prevalence of other parenterally-transmitted diseases, i.e. HBV and HCV The aims of the present study were therefore: i) to evaluate the prevalence of TTV infection in a sample of institutionalized elderly subjects; ii) to correlate TTV prevalence with HBV and HCV seroprevalence; iii) to correlate TTV prevalence with any underlying diseases and conditions. Material and methods STUDY POPULATION The study was carried out in an old people s nursing home in North-Eastern Italy (Veneto Region). All residents gave their informed consent to taking part in the study. The overall sample included 285 subjects (70 males, 215 females), whose mean age was 83.4 ± 9.1 years (range years). Subjects were interviewed whenever possible and/or their case-notes were examined. Sera were collected from all subjects and frozen at -80 C until they were analyzed. Laboratory methods Serum transaminases (alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma glutamyltransferase [ggt]) were assessed by standard methods. Any presence of antibodies against HCV (anti-hcv) was evaluated using commercially-available 3rd generation microparticle enzyme immunoassay (MEIA) version 3.0 (Axym System, Abbott Chicago IL, USA). HBsAg and anti-hbc were tested using an enzyme immunoassay (IMX System Abbott Diagnostics Labs, Chicago, IL, USA) according to the manufacturer s recommendations, including the specific confirmatory test. Anti-HCV positive sera were assayed for HCV-RNA by COBAS HCV Amplicor v2.0 Roche (Roche Molecular Systems, Inc, Branchbourg, NJ, USA), according 89

2 A. FLOREANI, V. BALDO, S. BUORO, ET AL. to the manufacturer s recommendations. HCV-RNApositive samples were typed by means of a commercial kit (INNOLiPA II line probe assay, Immunogenetics, Zwidarecht, Belgium) following the manufacturer s instructions. TTV-DNA detection DNA was extracted from 250 m l of serum by EX- TRAgene kit (Bioline Diagnostic, Torino, Italy) and resuspended in 30 m l water, according to the manufacturer s instructions. TTV-DNA was amplified by semi-nested PCR with TTV-specific primers compatible with different genotypes and subtypes as already published 3. Briefly, the first round of PCR was carried out with 0.5 m M NG059 primers (sense: 5 -ACA GAC AGA GGA GAA GGC AAC ATG-3 ) and NG063 (antisense: 5 -CTG GCA TTT TAC CAT TTC CAA AGT T-3 ) in 50 m l PCR mixture containing 5 m L of extracted DNA, 1x buffer, 0.2 mm dntps, 2 mm magnesium chloride, and 0.5U AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk CT, USA). Amplification was carried out with a 9600 Perkin Elmer Cetus DNA thermal cycler for 40 cycles (94 C, 30 s; 60 C, 45 s; 72 C, 45 s). The second-round PCR was performed with another sense primer NG061 (sense 5 -GCC AAC ATG YTR TGG ATA GAC TGG-3 [Y = T or C; R = A or G] and the same antisense primer NG063. One m l of the first round PCR was transferred to a new tube and amplified for 35 cycles in the same conditions as described above. After amplification, 10 m l of the PCR products were loaded onto 2% Nu-Sieve 3:1 agarose (FMC Bioproducts, Rockland, ME, USA), electrophoresed in 1x TBE buffer (89 mm Trisborate, 2 mm EDTA, ph 8.3), stained with ethidium bromide and photographed under ultraviolet light. The amplification products of the first-round PCR measured 286 base pairs (bp) and those of the second-round PCR measured 272 bp. STATISTICAL ANALYSIS Data analyses were performed by applying the chisquared test (Mantel Haenszel, Fisher s exact test and chi-squared for trend). A p value of 0.05 was considered significant, and an Odds Ratio (O.R.) with a 95% Confidence Interval (C.I.) was calculated for each parameter. Analyses were performed with the EPI-Info 6.04 computer program supplied by the Center for Disease Control and Prevention of Atlanta (GA, USA). Adjusted O.R. for each of the variables significantly associated with TTV infection were calculated using multiple logistic regression analysis with the Statistical Package for the Social Sciences (SPSS 8.5; Microsoft Corporation, Redmond/Washington, USA). Results Twenty-seven subjects (9.5%) were found TTV DNA positive. Females showed a higher prevalence of TTV DNA positivity than males, though the difference was not statistically significant [23/215 (10.7%) vs. 4/70 (5.7%), p = 0.217]. The highest rates of TTV positivity were found in the year-old age group (10.8%), while no correlation was found between TTV positivity and advancing age (c 2 for trend; p = 0.764). Univariate analysis showed that TTV infection did not correlate with the time spent at the nursing home; patients who were self-sufficient had a higher prevalence of TTV positivity, however, than subjects who were not (20.0% vs. 4.6%; OR: 5.17, CI: , p < 0.001). As far as associated diseases and conditions are concerned, subjects with diabetes exhibited the highest rate of TTV positivity (19.4%), though no statistical significant difference was observed in TTV prevalence between the different illness groups. Five of the 6 subjects with TTV infection and concomitant diabetes were insulin-dependent. HBsAg was tested in all 285 subjects: only one was found HBsAg positive and was not coinfected with TTV virus or HCV. Anti-HBc was tested in 119/285 subjects (41.8%): 12 (10.1%) were found antihbc positive; only 3 were found coinfected with TTV, but none was coinfected with HCV. Anti-HCV positivity was found in 33/285 subjects (11.6%) with a similar proportion in females and males (11.2% vs. 12.9%; p = 0.700). HCV-RNA was detected in 24 subjects (72.7%) and HCV genotyping showed type 1b in 17 (70.8%) and type 2a/2c in 7 (29.2%). Only 2 anti-hcv positive patients were coinfected with TTV. The highest rate of anti-hcv prevalence was found in the year-olds (10.8%). No correlation was found between anti-hcv positivity and advancing age, self-sufficiency, or TTV infection (Tab. I). Multivariate analysis showed that diabetes and selfsufficiency were independent factors associated with TTV positivity (p = and p = 0.001, respectively) (Tab. II). None of the subjects had altered liver function tests or evidence of liver disease. Discussion The results of our study indicate that institutionalized elderly subjects have a reasonable prevalence of TTV infection. Multivariate analysis showed that diabetes and self-sufficiency were independent factors associated with TTV positivity. TTV infection is not correlated with advancing age, gender, length of stay in the nursing home or concomitant HBV/HCV infection, however. It is worth stressing several points. First of all, the route of infection seems to differ from that of HCV or HBV. This suggests other than parenteral routes, possibly even the feco-oral. As far as HBV epidemiology in our geographical area is concerned, the main route is intrafamily spread 11 ; however, due to the strong decline in HBV infection in the last 10 years, we generally observe sporadic new events only in high-risk groups, such as drug addicts and homosexuals. In elderly peo- 90

3 TT VIRUS INFECTION IN THE ELDERLY Tab. I. Characteristics of the study group according to HCV positivity. n. HCV positive p Subgroup subjects n % Group of age < > Gender Males Females Self-sufficiency No Yes TTV positive No Yes Tab. II. Multivariate analysis according to TTV positivity. Characteristics O.R. C.I. p Age Diabetes Gender (females) Heart condition Lung conditions Self-sufficiency ple, the occasional infected subjects are generally longstanding HBsAg carriers whose HBV infection occurred in youth or early in their adult life. As far as HCV is concerned, several studies support a mainly epidemic spread in our geographical area after the Second World War, mainly due to the use of glass syringes for iv. and im. medication 10. We cannot say when TTV infection may have occurred, but our findings point to different risk factors from those relating to HBV or HCV. Multivariate analysis showed a surprising association with diabetes and selfsufficiency. The former association is consistent with Gallian et al. 12 finding of a high prevalence of TTV-infected subjects among hemodialysis patients and also in a population of patients with diabetes but without renal disease. In the French study, insulin-dependent diabetic patients were not distinguished from those who were not, but in our study TTV infection was strongly associated with insulin-dependent diabetes. We could only suggest that patients with diabetes are more often involved in clinical examinations, hospitalization, and the medical treatment of several complications than non-diabetics. The risk of parenterally-transmitted viruses might consequently be much higher. On the other hand, the finding of a statistically significant association with self-sufficiency is apparently in contrast with such a hypothesis. It is likely that at least some of the TTV-positive subjects acquired the infection earlier in their lives. The reported prevalence of TTV infection varies considerably in different geographical areas. It is reasonable to consider several factors which might explain the different epidemiology, including the selection of blood donors (whether or not they are remunerated, previous history of exposure to blood products, selection of subjects from the general population, etc.). The major drawback of our study is the restriction to NG059/63-61 primer set which only indicates present infection. However, using the same primers Fabris et al. 13 found that the rate of TTV infection is 16.6% in patients with non-a-e acute hepatitis and 6.6% in healthy controls with a mean age of 38 years. The 9.5% rate observed in the present study is therefore consistent with a medium-grade spread of TTV infection in the general population. Obviously, future studies are warranted to establish the length of viremia and the relationship between TTV infection and immune response. Finally, our study confirms the lack of any association with overt liver disease. In conclusion, TTV infection is also frequent in the elderly, though in lower percentages than HCV infection. The infection is not correlated with HCV and HBV routes and the main risk factors in the elderly seem to be an association with diabetes and self-sufficiency. Due to apparently contrasting data on the epidemiology of TTV infection around the world, the hypothesis of TTV s non-pathogenicity requires careful re-consideration based upon long-term, extensive prospective studies. 91

4 A. FLOREANI, V. BALDO, S. BUORO, ET AL. References 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: Viazov S, Ross RS, Niel C, de Oliveira JM, Varenholz C, Da Villa G, Roggendorf M. Sequence variability in the putative coding region of TT virus: evidence of two rather than several major types. J Gen Virol 1998;79: Okamoto H, Nishizawa T, Kato N, Ukita M, Ikeda H, Iizuka H, Miyakawa Y, Mayumi M. Molecular cloning and characterisation of a novel DNA virus (TTV) associated with posttrasfusion hepatitis of unknown etiology. Hepatol Res 1998;10: Simmonds P, Davindson F, Lycett C, Prescott DM, Ellender J, Yap PL, Ludlam CA, Haydon GH, Gillon J, Jarvis LM. Detection of a novel DNA virus (TTV) in blood donors and blood products. Lancet 1998;352: MacDonald DM, Scott GR, Clutterbuck D, Simmonds P. Infrequent detection of TTV Virus Infection in intravenous drug users prostitutes and homosexual men. J Infect Dis 1999;179: Okamoto H, Akahane Y, Ukita M, Fukuda M, Tsuda F, Miyakawa Y, Mayumi M. Fecal excretion of a nonenvepoled DNA virus (TTV) Associated with posttransfusion Non-A-G hepatitis. J Med Virol 1998;156: Charlton M, Adjei P, Poterucha J, Zein N, Moore B, Therneau T, Krom R, Wiesner R. TT-virus Infection in North America Blood Donors Patients with fulminate Hepatic failure and cryptogenic Cirrhosis. Hepatology 1998;28: Stroffolini T, Menchinelli M, Taliani G, Stroffolini T, Menchinelli M, Taliani G, Dambruoso V, Poliandri G, Bozza A, Lecce R, Clementi C, Ippolito FM, Compagnoni A. High prevalence of hepatitis C virus infection in a small central Italian town: lack of evidence of parenteral exposure. Ital J Gastroenterol 1995;27: Guadagnino V, Stroffolini T, Rapicetta M, Costantino A, Kondili LA, Menniti-Ippolito F, Caroleo B, Costa C, Griffo C, Loiacono L, Pisani V, Foca A, Piazza M. Prevalence risk factors and molecular epidemiology of hepatitis C virus infection in the general population. A community-based survey in Southern Italian town. Hepatology 1997;26: Chiaramonte M, Stroffolini T, Lorenzoni U, Minniti F, Conti S, Floreani A, Ntakirutimana E, Vian A, Ngatchu T, Naccarato R. Risk factors in community-acquired chronic hepatitis C virus infection: a case-control study in Italy. J Hepatol 1996;24: Chiaramonte M, Trivello R, Stroffolini T, Rapicetta M, Bertin T, Renzulli G, Ngatchu T, Chionne P, Trivello R, Naccarato R. Changing pattern of hepatitis B infection in children: a comparative seroepidemiological study (1979 vs 1989) in north-east Italy. Ital J Gastroenterol 1991;23: Gallian P, Berland Y, Olmer M, Raccah D, de Micco P, Biagini P, Simon S, Bouchouareb D, Mourey C, Roubicek C, Touinssi M, Cantaloube JF, Dussol B, de Lamballerie X. TT virus infection in French hemodialysis patients: study of prevalence and risk factors. J Clin Microbiol 1999;37: Fabris P, Biasin MR, Infantolino D, Tositti G, Venza E, Floreani A, Zanetti A, de Lalla F. TTV infection in patients with acute hepatitis of defined etiology and in non-a-e hepatitis. J Hepatol 2000;32: This work was partially supported by a university grant (MURST 60%). Correspondence: dr. Vincenzo Baldo, Department of Environmental Medicine and Public Health, Institute of Hygiene, via Loredan 18, Padua (Italy) - Tel Fax vbaldo@ux1.unipd.it 92

5 93 TTV VIRUS INFECTION IN THE ELDERLY