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The molecular epidemiology and clinical significance of TT virus (TTV) infection in healthy blood donors from Southern Taiwan
Transfusion and Apheresis Science 24 (2001) 9±15
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The molecular epidemiology and clinical signi®cance of TT virus (TTV) infection in healthy blood donors from Southern Taiwan Chia-Yen Dai a, Ming-Lung Yu a, Wan-Long Chuang a, Ching-Shan Wang b, Zu-Yau Lin a, Shinn-Cherng Chen a, Ming-Yuh Hsieh a, Liang-Yen Wang a, Jung-Fa Tsai a, Wen-Yu Chang a,* a
Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC b Kaohsiung Blood Center, Chinese Blood Services Foundation, Taiwan, ROC Received 2 August 2000; accepted 13 September 2000
Abstract To evaluate the molecular epidemiology and clinical signi®cance of the TT virus (TTV) infection in healthy blood donors from southern Taiwan, 475 sera of healthy volunteer blood donors were tested for: TTV DNA, GB virus C/ hepatitis G virus (GBV-C/HGV) anti-envelope protein 2 antibody and RNA. TTV DNA was positive in 111 donors (23.4%). Age and the rate of elevated ALT levels were signi®cantly higher in TTV viremic donors than TTV non-viremic donors (P < 0:01, P < 0:05). Phylogenetic analysis of 49 donors revealed 22 (44.9%), 13 (32.7%), 1 (2%), 10 (20.4%), and 3 (6.1%) Taiwanese TTV isolates clustered in genotypes 1a, 1b, 2a, 2b, and 4, respectively. By multivariate analyses, age and elevated ALT levels were signi®cant factors associated with TTV viremia. In conclusion, TTV infection, predominant genotype 1, is highly prevalent in Taiwanese blood donors. TTV viremia is associated with increased age and elevated ALT levels. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: TT virus DNA; GBV-C/HGV RNA; Blood donor
1. Introduction To avoid the transmission of hepatitis agents such as hepatitis B virus (HBV) and hepatitis C virus (HCV), screening tests have been introduced into the blood banks. Nevertheless, sporadic cases
* Corresponding author. Tel.: +886-7-3121101 ext. 6014; fax: +886-7-3118141. E-mail address: [email protected] (W.-Y. Chang).
of posttransfusion hepatitis are still observed. More eorts have been made to identify new agents responsible for these patients and potential causative viral agents were discovered and investigated subsequently. A transfusion-transmissible ¯avivirus-like virus named GB virus C (GBV-C) and another isolate of GBV-C, hepatitis G virus (GBV-C/HGV) have been isolated [1,2] and claimed to be associated with posttransfusion [2], acute community-acquired [1,2], fulminant or chronic [3], and even
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hepatocellular carcinoma [4]. However, other reports have indicated that GBV-C/HGV does not account for liver diseases [5±7]. The pathogenicity and hepatopathic eects of GBV-C/HGV infection remain controversial. In 1997, a DNA was cloned from an acute phase serum of a Japanese patient with posttransfusion hepatitis of unknown etiology [8]. The clone was derived from a novel non-enveloped, single-stranded DNA virus [8,9] which was designated TT virus (TTV) after the initials (T.T.) of the index patient [9]. Initially, TTV was considered to have a linear genome, with 3739 bases and two opening reading frames encoding 770 and 150 aminoacids, and similar to Parvoviruses [9]. Further molecular studies showed that TTV, with circular genome which comprises 3852 bases with a particle size of 30±50 nm, resembles the circoviridae [10]. The prevalence of TTV in blood donors around the world varies from 1.9% in Scotland [11] to 81.7% reported in Japan [12]. The clinical signi®cance of TTV infection is still far from de®nite. There were some reports suggesting that TTV may be associated with elevated ALT levels [8,9,13±15] and may play a role in the development of chronic liver diseases of unknown etiology [8,16,17] or even hepatocellular carcinoma [18]. In contrast, numerous investigations revealed that the presence of TTV DNA was not correlated with abnormal ALT levels [19±21], liver injury [22±24] or hepatocellular carcinoma [12]. With a wide range of sequence divergence, TTV was classi®ed into genotypes (1 and 2) which were further divided into subtypes (a and b) [9]. Recent analyses, using two dierent strategies for molecular phylogenetic analysis, resulted in six genotypes of TTV being obtained [25]. A relationship between TTV genotypes and liver diseases was not found. To evaluate the prevalence of TTV in blood donors from southern Taiwan, a polymerase chain reaction (PCR) method was used to detect the presence of TTV DNA. Phylogenetic analysis was also performed. The clinical factors associated with TTV infection and the possible relationship between TTV and GBV-C/HGV, thought to be transfusion-transmissible viruses, were investigated.
2. Methods 2.1. Subjects Four hundred and seventy-®ve volunteer blood donors from a blood center in Kaohsiung, the largest city in southern Taiwan, were enrolled in the study. There were 368 men and 107 women ranging in age from 18 to 50 years (mean standard deviation [S.D.]: 28.9 8.8 years). All the serum samples were negative for both HBsAg and Anti-HCV. 2.2. Laboratory tests Second-generation HCV antibody and HBsAg were detected with commercially available enzymelinked immunosorbent assay (ELISA) kits (Abbott, North Chicago, IL). Alanine aminotransferase (ALT) (normal upper limit of serum ALT 25 IU/l) was measured on a multichannel autoanalyzer. 2.3. Detection of TTV DNA in serum TTV DNA was detected by nested PCR using semi-nested primers targeting the N22 region in the coding region as described previously [9]. Brie¯y, Total DNA was puri®ed from 150 ll serum using the QIAamp Blood Kit (Qiagen, Hilden, Germany) and eluted in a ®nal volume of 50 ll. Five ll were added to the ®rst round of PCR with the sense primer NG059 (50 -ACA GAC AGA GGA GAA GGC AAC ATG-30 ) and the antisense primer NG063 (50 -CTG GCA TTT TAC CAT TTC CAA AGT T-30 ) for 5 min at 95°C, followed by 35 cycles consisting of denaturation for 30 s at 94°C, annealing for 45 s at 60°C, and extension for 45 s at 72°C (additional 7 min for the last cycle). The second round of PCR was performed with the sense primer NG061 (50 -GGC AAC ATG YTR TGG ATA GAC TGG-30 Y T or C; R A or G) and the anti-sense primer NG063 for 25 cycles under the same conditions as used for the ®rst round of PCR. The ampli®cation products by the ®rst and the second round PCR measured 286 and 271 base pairs. PCR products were analyzed by gel electrophoresis with 3% agarose, stained with ethidium bromide and observed under ultraviolet light.
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2.4. Nucleotide sequencing and phylogenetic analysis of TTV DNA Both strands of DNA from nested PCR products, excluding the primers, were puri®ed by QIAquick PCR puri®cation kit (Qiagen) and directly sequenced from both directions using an ABI PRISMe BigDyee Terminator Cycle Sequencing Ready Reaction (Perkin Elmer Cetus, Norwalk, CT) with an ABI 310 Genetic Analyzer (Applied Biosystems). Sequence comparison and phylogenetic analyses were done with the aid of computer software, using the Clustal method with a weighted-residue weight table (DNAstar). 2.5. Detection of GBV-C/HGV RNA and anti-E2 antibody in serum GBV-C/HGV RNA was detected by nested reverse transcription PCR (RT-PCR) using primers targeting the 50 UTR as described previously [26]. The anti-E2 antibody was measured by an enzyme linked immunosorbent assay from Boehringer Mannheim (GmbH, Germany), strictly according to the manufacturer's instructions [27]. 2.6. Statistical analyses Descriptive statistics such as means and proportions were calculated. Frequency was compared between groups using the Chi-square test or Fisher's exact test, and group means were compared using the t-test. Contingency table analysis (Chi-squared test for trend) was carried out to demonstrate the increase of TTV seroprevalence with age. The presence of a statistical signi®cance was inferred when P was less than 0.05. According to the textbook of statistics (Beth-Dawson-Saunders and Robert G. Trapp. Basic and Clinical Biostatistics, Englewood Clis: Prentic-Hall, 1990), the P value less than 0.05 is considered to be statistically signi®cant. Therefore, we used the level of 0.05 as the threshold of signi®cance in our study. Stepwise logistic regression method was used to analyze the study data. Odds ratios (ORs) and their associated 95% con®dence intervals (CIs) were used to quantify the magnitude of their associations.
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3. Results 3.1. TTV viremia Of 475 blood donors aged 18±50 years without HBsAg and Anti-HCV, 111 were positive for TTV DNA, showing an overall prevalence of 23.4%. The clinical characteristics between individuals with and without TTV DNA were analyzed and are shown in Table 1. No signi®cant dierence was observed between TTV infection rates among male (90/368, 24.5%) and female (21/107, 19.6%) donors. The mean ages of the 111 positive individuals were signi®cantly dierent from those of 364 negative ones (31.2 9.7 vs. 28.2 8.4 years, P < 0:01). In addition, stratifying subjects by three age groups, 18±20, 21±35, and 36±50 years old, revealed signi®cant dierence in the distributions of the TTV DNA among the three age groups (P < 0:05). TTV DNA was found in 7 (14.9%), 68 (21.9%) and 36 (31.6%) of the three age groups, respectively. 3.2. GBV-C/HGV viremia, anti-E2 antibodies and TTV viremia GBV-C/HGV RNA was present in 14 (2.9%) of the studied samples. With 33 (6.9%) positive for anti-E2 antibodies and 2 (0.4%) positive for both serum GBV-C/HGV RNA and anti-E2 antibodies, the prevalence of HGV exposure, de®ned as positive for serum GBV-C/HGV RNA and/or anti-E2, was 9.5%. There was no association between the presence of GBV-C/HGV RNA and TTV DNA or between the GBV-C/HGV exposure rate and TTV DNA. 3.3. Serum ALT levels and TTV viremia The mean serum ALT levels of the studied samples was 14.7 11.1 IU/l. Serum ALT levels in the 111 subjects positive for TTV DNA were signi®cantly higher than those of the 364 subjects negative for TTV DNA (16.7 13.3 vs. 14.1 10.1 IU/l, P < 0:05). In addition, signi®cant difference was observed in the rates for ALT level abnormality (>25 IU/l) between those positive for
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Table 1 Comparison of clinical characteristics between individuals with and without TTV viremia in blood donors No
No (%) of patients with TTV DNA Positive
Negative
Sex Male Female
368 107
90 (24.5) 21 (19.6)
278 (75.5) 86 (80.4)
Age (year, mean S.D.)a <20 >20, <35 >35
47 314 114
31.2 9.7 7 (14.9) 68 (21.7) 36 (31.6)
28.2 8.4 40 (85.1) 246 (78.3) 78 (68.4)
ALT (IU/l, mean S.D.)b Normal (625 IU/l) Abnormal (>25 IU/l)
423 52
16.7 13.3 92 (21.8) 19 (36.5)
14.1 10.1 331 (78.2) 33 (63.5)
<0.05 <0.05
GBV-C/HGV exposurec Positive Negative
45 430
8 (17.8) 103 (23.9)
37 (82.2) 327 (76.1)
NS
GBV-C/HGV RNA Positive Negative
14 461
3 (21.4) 108 (23.4)
11 (78.6) 353 (76.6)
NS
Blood donation First Repeat
79 396
13 (16.5) 98 (24.8)
66 (83.5) 298 (75.2)
NS
P NS
<0.01 <0.05
a
Mean S.D.: mean standard deviation. ALT: alanine aminotransferase. c GBV-C/HGV exposure: positive for GBV-C/HGV RNA and/or anti-E2 antibodies. b
TTV DNA (19/111, 17.1%) and those negative for TTV DNA (33/364, 9.1%) (P < 0:05). 3.4. Times of blood donation and TTV viremia Seventy-nine of the 475 blood donors (16.7%) donated blood for the ®rst time. When the TTV DNA positive rates between individuals with ®rst or repeated donations were compared, no signi®cant dierence was observed (16.5 vs. 24.8%, respectively). 3.5. Sequencing and phylogenetic analysis of TTV DNA in TTV viremic patients TTV DNA genomes from 49 donors were sequenced directly and phylogenetic analysis was carried out. The results showed that in regard to the subtypes of TTV, 22 (44.9%) were more closely related to the genotype 1a, 13 (32.7%) to genotype
1b, 1 (2%) to genotype 2a, 10 (20.4%) to genotype 2b, and 3 (6.1%) to genotype 4. Total 77.6% of TTV isolates of blood donors were deduced to be TTV type 1. 3.6. Serum ALT levels and TTV genotypes Elevated ALT level was found in 11 sera with 6 (27.3%) of type 1a, 2 (15.4%) of type 1b, and 3 (25%) of non-type 1 cases. No signi®cant dierence was observed in the rates for ALT level abnormality between genotype 1 and non-genotype 1 individuals. 3.7. Factors associated with TTV viremia Based on the results of multiple logistic regression analysis of positive TTV DNA, two factors including increased age and abnormal ALT levels were identi®ed to be independently associated with
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Table 2 Stepwise logistic regression analysis of factors signi®cantly associated with TTV DNA positivity in blood donors Dependent variable TTV DNA Positive
a
Independent variable
Comparison
Odds ratio (95% C.I.a )
Age ALT
Each year-old increase Abnormal vs. normal
1.03 (1.01±1.06) 1.9 (1.02±3.52)
C.I.: con®dence interval.
positive TTV DNA, as odds ratios and their corresponded 95% con®dence intervals are shown in Table 2. 4. Discussion The global distribution of TTV has been documented with a great variety of prevalence rates in dierent countries. TTV viremia can be detected by PCR methods using primers deduced from N22 region [9]. The TTV prevalences in blood donors by dierent primers were 1.9% in UK [11], 12± 81.7% in Japan [9,12,24], 62% in Brazil [20], 28% in China [28], and 1% in North America [29]. We report here that the prevalence of TTV DNA by N22 region primers in the southern Taiwanese blood donors negative for HBsAg and Anti-HCV is 23.4%. This result is higher than 11% from eastern Taiwan aboringines [19] and between 10% and 53% from northern Taiwan healthy subjects [22,23]. The clinical implication of TTV infection and etiological importance of TTV in association with liver diseases still remain unclear. After excluding the in¯uence of HBV or HCV infection, our study indicated that elevated ALT levels were associated with TTV infection. With the initial ®nding of TTV infection in posttransfusion hepatitis [8], TTV infection was suggested to cause hepatitis with elevated ALT levels [8,16,17]. Nevertheless, other reports revealed TTV infected subjects had normal ALT levels [19±21]. These dierent results may be explained by the possibility of a healthy carrier state of TTV infection [21]. Besides, when we consider the association between TTV infection and elevated ALT levels, two possible explanations are made: (1) TTV may really cause abnormality of liver function test, (2) people with elevated ALT
levels may be under the special circumstances and have higher rates of TTV infection. The prevalence of GBV-C/HGV RNA in our volunteer donors was 2.9% which was similar to the other reports of 0.9±3% in dierent parts of the world [2,30±32] and reports of 2.1% from Taiwan [33]. In our study, TTV viremia failed to show association with HGV exposure or viremia. We attributed the results to the lack of recovery markers for TTV infection to indicate the real exposure of TTV as anti-E2 in GBV-C/HGV infection [34,35]. The transmission of GBV-C/HGV was mainly through high-risk procedures or lifestyles, such as blood transfusion [2,36], intravenous drug use or sexual contact [37]. Our results may also indicate other important transmission routes dierent from GBV-C/HGV, such as the fecal-oral route in TTV infection [38,39]. There is a gradual increase in TTV infection rates with age in TTV infection groups shown in our study. With the similar previous reports for newborn to aged population [22,39], our results support that TTV has non-parenteral transmission routes and there is persistent TTV viremia [21]. Repeated donations did not decrease the TTV viremia rate in blood donors. This depicts the fact that available screening tests nowadays for donated blood cannot prevent TTV infection entirely. In the present study, the genetic heterogeneity of isolates cloned from southern Taiwanese donors existed by phylogenetic analysis of nucleotide sequence from the TTV genome. TTV genotype 1, which is distributed widely and most prevalent over the world, is also the most prevalent type among TTV isolates in our study as in previous reports from northern Taiwan [23] and Japan [24]. Genotype 1 had been reported to be associated with more pathogenetic potential [29]. We did not ®nd a signi®cant relationship between abnormal
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ALT levels and TTV genotypes as in other reports [23±25]. Further investigation will be required to de®ne the pathogenicity and virological characteristics of TTV. References [1] Simons JN, Leary TP, Dawson GJ, Pilot-Matias TJ, Muerho AS, Schlauder GG et al. Isolation of novel viruslike sequences associated with human hepatitis. Nat Med 1995;1:564±9. [2] Linnen J, Wages Jr. J, Zhang-Keck ZY, Fry KE, Krawczynski KZ, Alter H et al. Molecular cloning and disease association of hepatitis G virus: a transfusiontransmissible agent. Science 1996;271:505±8. [3] Yoshiba M, Okamoto H, Mishiro S. Detection of the GBV-C hepatitis virus genome in serum from patients with fulminant hepatitis of unknown etiology. Lancet 1995;346:1131±2. [4] Tagger A, Donato F, Ribero ML, Chiesa R, Tomasoni V, Portera G et al. A case-control study on GB virus C/ hepatitis G virus infection and hepatocellular carcinoma. Brescia HCC study. Hepatology 1997;26:1653±7. [5] Alter MJ, Gallagher M, Morris TT, Moyer LA, Meeks EL, Krawczynski K et al. Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection. N Engl J Med 1997;336:741±6. [6] Alter HJ, Nakatsuji Y, Melpolder J, Wages J, Wesley R, Shih JW et al. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. N Engl J Med 1997;336:747±54. [7] Leary TP, Muerho AS, Simons JN, Pilot-Matias TJ, Erker JC, Chalmers ML et al. Sequence and genomic organization of GBV-C: a novel member of the ¯aviviridae associated with human non-A-E hepatitis. J Med Virol 1996;48:60±7. [8] 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±7. [9] Okamoto H, Nishizawa T, Kato N, Ukita M, Ikeda H, Iizuka H et al. Molecular cloning and characterization of a novel virus (TTV) associated with posttransfusion hepatitis of unknown etiology. Hepatol Res 1998;10:1±16. [10] Mushahwar IK, Erker JC, Muerho AS, Leary TP, Simons JN, Birkenmeyer LG et al. Molecular and biophysical characterization of TT virus: evidence for a new virus family infecting humans. Proc Natl Acad Sci USA 1999;96:3177±82. [11] Simmonds P, Davidson F, Lycett C, Prescott LE, MacDonald DM, Ellender J et al. Detection of a novel DNA virus (TTV) in blood donors and blood products. Lancet 1998;352:191±5.
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[33] Wang JT, Chen PJ, Liu DP, Sheu JC, Wang TH, Chen DS. Prevalence and infectivity of hepatitis G virus and its strain variant, the GB agent, in volunteer blood donors in Taiwan. Transfusion 1998;38:290±5. [34] Tacke M, Kiyosawa K, Stark K, Schlueter V, OfenlochHaehnle B, Hess G et al. Detection of antibodies to a putative hepatitis G virus envelope protein. Lancet 1997;349:318±20. [35] Pilot-Matias TJ, Carrick RJ, Coleman PF, Leary TP, Surowy TK, Simons JN et al. Expression of the GB virus C E2 glycoprotein using the Semliki Forest virus vector system and its utility as a serologic marker. Virology 1996;225:282±92. [36] Hsu SC, Chang MH, Ni YH, Hsu HY, Lee CY. Horizontal transmission of hepatitis B virus in children. J Pediat Nutr 1993;16:66±9. [37] Hurie MB, Mast EE, Davis JP. Horizontal transmission of hepatitis B virus infection to United States-born children of Hmong refugees. Pediatrics 1992;89:269±73. [38] Okamoto H, Akahane Y, Ukita M, Fukuda M, Tsuda F, Miyakawa Y et al. Fecal excretion of a non-enveloped DNA virus (TTV) associated with posttransfusion nonA-G hepatitis. J Med Virol 1998;56:128±32. [39] Saback FL, Gomes SA, de Paula VS, da Silva RR, LewisXimenez LL, Niel C. Age-speci®c prevalence and transmission of TT virus. J Med Virol 1999;59:318±22.
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The molecular epidemiology and clinical signi®cance of TT virus (TTV) infection in healthy blood donors from Southern Taiwan Chia-Yen Dai a, Ming-Lung Yu a, Wan-Long Chuang a, Ching-Shan Wang b, Zu-Yau Lin a, Shinn-Cherng Chen a, Ming-Yuh Hsieh a, Liang-Yen Wang a, Jung-Fa Tsai a, Wen-Yu Chang a,* a
Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC b Kaohsiung Blood Center, Chinese Blood Services Foundation, Taiwan, ROC Received 2 August 2000; accepted 13 September 2000
Abstract To evaluate the molecular epidemiology and clinical signi®cance of the TT virus (TTV) infection in healthy blood donors from southern Taiwan, 475 sera of healthy volunteer blood donors were tested for: TTV DNA, GB virus C/ hepatitis G virus (GBV-C/HGV) anti-envelope protein 2 antibody and RNA. TTV DNA was positive in 111 donors (23.4%). Age and the rate of elevated ALT levels were signi®cantly higher in TTV viremic donors than TTV non-viremic donors (P < 0:01, P < 0:05). Phylogenetic analysis of 49 donors revealed 22 (44.9%), 13 (32.7%), 1 (2%), 10 (20.4%), and 3 (6.1%) Taiwanese TTV isolates clustered in genotypes 1a, 1b, 2a, 2b, and 4, respectively. By multivariate analyses, age and elevated ALT levels were signi®cant factors associated with TTV viremia. In conclusion, TTV infection, predominant genotype 1, is highly prevalent in Taiwanese blood donors. TTV viremia is associated with increased age and elevated ALT levels. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: TT virus DNA; GBV-C/HGV RNA; Blood donor
1. Introduction To avoid the transmission of hepatitis agents such as hepatitis B virus (HBV) and hepatitis C virus (HCV), screening tests have been introduced into the blood banks. Nevertheless, sporadic cases
* Corresponding author. Tel.: +886-7-3121101 ext. 6014; fax: +886-7-3118141. E-mail address: [email protected] (W.-Y. Chang).
of posttransfusion hepatitis are still observed. More eorts have been made to identify new agents responsible for these patients and potential causative viral agents were discovered and investigated subsequently. A transfusion-transmissible ¯avivirus-like virus named GB virus C (GBV-C) and another isolate of GBV-C, hepatitis G virus (GBV-C/HGV) have been isolated [1,2] and claimed to be associated with posttransfusion [2], acute community-acquired [1,2], fulminant or chronic [3], and even
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C.-Y. Dai et al. / Transfusion and Apheresis Science 24 (2001) 9±15
hepatocellular carcinoma [4]. However, other reports have indicated that GBV-C/HGV does not account for liver diseases [5±7]. The pathogenicity and hepatopathic eects of GBV-C/HGV infection remain controversial. In 1997, a DNA was cloned from an acute phase serum of a Japanese patient with posttransfusion hepatitis of unknown etiology [8]. The clone was derived from a novel non-enveloped, single-stranded DNA virus [8,9] which was designated TT virus (TTV) after the initials (T.T.) of the index patient [9]. Initially, TTV was considered to have a linear genome, with 3739 bases and two opening reading frames encoding 770 and 150 aminoacids, and similar to Parvoviruses [9]. Further molecular studies showed that TTV, with circular genome which comprises 3852 bases with a particle size of 30±50 nm, resembles the circoviridae [10]. The prevalence of TTV in blood donors around the world varies from 1.9% in Scotland [11] to 81.7% reported in Japan [12]. The clinical signi®cance of TTV infection is still far from de®nite. There were some reports suggesting that TTV may be associated with elevated ALT levels [8,9,13±15] and may play a role in the development of chronic liver diseases of unknown etiology [8,16,17] or even hepatocellular carcinoma [18]. In contrast, numerous investigations revealed that the presence of TTV DNA was not correlated with abnormal ALT levels [19±21], liver injury [22±24] or hepatocellular carcinoma [12]. With a wide range of sequence divergence, TTV was classi®ed into genotypes (1 and 2) which were further divided into subtypes (a and b) [9]. Recent analyses, using two dierent strategies for molecular phylogenetic analysis, resulted in six genotypes of TTV being obtained [25]. A relationship between TTV genotypes and liver diseases was not found. To evaluate the prevalence of TTV in blood donors from southern Taiwan, a polymerase chain reaction (PCR) method was used to detect the presence of TTV DNA. Phylogenetic analysis was also performed. The clinical factors associated with TTV infection and the possible relationship between TTV and GBV-C/HGV, thought to be transfusion-transmissible viruses, were investigated.
2. Methods 2.1. Subjects Four hundred and seventy-®ve volunteer blood donors from a blood center in Kaohsiung, the largest city in southern Taiwan, were enrolled in the study. There were 368 men and 107 women ranging in age from 18 to 50 years (mean standard deviation [S.D.]: 28.9 8.8 years). All the serum samples were negative for both HBsAg and Anti-HCV. 2.2. Laboratory tests Second-generation HCV antibody and HBsAg were detected with commercially available enzymelinked immunosorbent assay (ELISA) kits (Abbott, North Chicago, IL). Alanine aminotransferase (ALT) (normal upper limit of serum ALT 25 IU/l) was measured on a multichannel autoanalyzer. 2.3. Detection of TTV DNA in serum TTV DNA was detected by nested PCR using semi-nested primers targeting the N22 region in the coding region as described previously [9]. Brie¯y, Total DNA was puri®ed from 150 ll serum using the QIAamp Blood Kit (Qiagen, Hilden, Germany) and eluted in a ®nal volume of 50 ll. Five ll were added to the ®rst round of PCR with the sense primer NG059 (50 -ACA GAC AGA GGA GAA GGC AAC ATG-30 ) and the antisense primer NG063 (50 -CTG GCA TTT TAC CAT TTC CAA AGT T-30 ) for 5 min at 95°C, followed by 35 cycles consisting of denaturation for 30 s at 94°C, annealing for 45 s at 60°C, and extension for 45 s at 72°C (additional 7 min for the last cycle). The second round of PCR was performed with the sense primer NG061 (50 -GGC AAC ATG YTR TGG ATA GAC TGG-30 Y T or C; R A or G) and the anti-sense primer NG063 for 25 cycles under the same conditions as used for the ®rst round of PCR. The ampli®cation products by the ®rst and the second round PCR measured 286 and 271 base pairs. PCR products were analyzed by gel electrophoresis with 3% agarose, stained with ethidium bromide and observed under ultraviolet light.
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2.4. Nucleotide sequencing and phylogenetic analysis of TTV DNA Both strands of DNA from nested PCR products, excluding the primers, were puri®ed by QIAquick PCR puri®cation kit (Qiagen) and directly sequenced from both directions using an ABI PRISMe BigDyee Terminator Cycle Sequencing Ready Reaction (Perkin Elmer Cetus, Norwalk, CT) with an ABI 310 Genetic Analyzer (Applied Biosystems). Sequence comparison and phylogenetic analyses were done with the aid of computer software, using the Clustal method with a weighted-residue weight table (DNAstar). 2.5. Detection of GBV-C/HGV RNA and anti-E2 antibody in serum GBV-C/HGV RNA was detected by nested reverse transcription PCR (RT-PCR) using primers targeting the 50 UTR as described previously [26]. The anti-E2 antibody was measured by an enzyme linked immunosorbent assay from Boehringer Mannheim (GmbH, Germany), strictly according to the manufacturer's instructions [27]. 2.6. Statistical analyses Descriptive statistics such as means and proportions were calculated. Frequency was compared between groups using the Chi-square test or Fisher's exact test, and group means were compared using the t-test. Contingency table analysis (Chi-squared test for trend) was carried out to demonstrate the increase of TTV seroprevalence with age. The presence of a statistical signi®cance was inferred when P was less than 0.05. According to the textbook of statistics (Beth-Dawson-Saunders and Robert G. Trapp. Basic and Clinical Biostatistics, Englewood Clis: Prentic-Hall, 1990), the P value less than 0.05 is considered to be statistically signi®cant. Therefore, we used the level of 0.05 as the threshold of signi®cance in our study. Stepwise logistic regression method was used to analyze the study data. Odds ratios (ORs) and their associated 95% con®dence intervals (CIs) were used to quantify the magnitude of their associations.
11
3. Results 3.1. TTV viremia Of 475 blood donors aged 18±50 years without HBsAg and Anti-HCV, 111 were positive for TTV DNA, showing an overall prevalence of 23.4%. The clinical characteristics between individuals with and without TTV DNA were analyzed and are shown in Table 1. No signi®cant dierence was observed between TTV infection rates among male (90/368, 24.5%) and female (21/107, 19.6%) donors. The mean ages of the 111 positive individuals were signi®cantly dierent from those of 364 negative ones (31.2 9.7 vs. 28.2 8.4 years, P < 0:01). In addition, stratifying subjects by three age groups, 18±20, 21±35, and 36±50 years old, revealed signi®cant dierence in the distributions of the TTV DNA among the three age groups (P < 0:05). TTV DNA was found in 7 (14.9%), 68 (21.9%) and 36 (31.6%) of the three age groups, respectively. 3.2. GBV-C/HGV viremia, anti-E2 antibodies and TTV viremia GBV-C/HGV RNA was present in 14 (2.9%) of the studied samples. With 33 (6.9%) positive for anti-E2 antibodies and 2 (0.4%) positive for both serum GBV-C/HGV RNA and anti-E2 antibodies, the prevalence of HGV exposure, de®ned as positive for serum GBV-C/HGV RNA and/or anti-E2, was 9.5%. There was no association between the presence of GBV-C/HGV RNA and TTV DNA or between the GBV-C/HGV exposure rate and TTV DNA. 3.3. Serum ALT levels and TTV viremia The mean serum ALT levels of the studied samples was 14.7 11.1 IU/l. Serum ALT levels in the 111 subjects positive for TTV DNA were signi®cantly higher than those of the 364 subjects negative for TTV DNA (16.7 13.3 vs. 14.1 10.1 IU/l, P < 0:05). In addition, signi®cant difference was observed in the rates for ALT level abnormality (>25 IU/l) between those positive for
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Table 1 Comparison of clinical characteristics between individuals with and without TTV viremia in blood donors No
No (%) of patients with TTV DNA Positive
Negative
Sex Male Female
368 107
90 (24.5) 21 (19.6)
278 (75.5) 86 (80.4)
Age (year, mean S.D.)a <20 >20, <35 >35
47 314 114
31.2 9.7 7 (14.9) 68 (21.7) 36 (31.6)
28.2 8.4 40 (85.1) 246 (78.3) 78 (68.4)
ALT (IU/l, mean S.D.)b Normal (625 IU/l) Abnormal (>25 IU/l)
423 52
16.7 13.3 92 (21.8) 19 (36.5)
14.1 10.1 331 (78.2) 33 (63.5)
<0.05 <0.05
GBV-C/HGV exposurec Positive Negative
45 430
8 (17.8) 103 (23.9)
37 (82.2) 327 (76.1)
NS
GBV-C/HGV RNA Positive Negative
14 461
3 (21.4) 108 (23.4)
11 (78.6) 353 (76.6)
NS
Blood donation First Repeat
79 396
13 (16.5) 98 (24.8)
66 (83.5) 298 (75.2)
NS
P NS
<0.01 <0.05
a
Mean S.D.: mean standard deviation. ALT: alanine aminotransferase. c GBV-C/HGV exposure: positive for GBV-C/HGV RNA and/or anti-E2 antibodies. b
TTV DNA (19/111, 17.1%) and those negative for TTV DNA (33/364, 9.1%) (P < 0:05). 3.4. Times of blood donation and TTV viremia Seventy-nine of the 475 blood donors (16.7%) donated blood for the ®rst time. When the TTV DNA positive rates between individuals with ®rst or repeated donations were compared, no signi®cant dierence was observed (16.5 vs. 24.8%, respectively). 3.5. Sequencing and phylogenetic analysis of TTV DNA in TTV viremic patients TTV DNA genomes from 49 donors were sequenced directly and phylogenetic analysis was carried out. The results showed that in regard to the subtypes of TTV, 22 (44.9%) were more closely related to the genotype 1a, 13 (32.7%) to genotype
1b, 1 (2%) to genotype 2a, 10 (20.4%) to genotype 2b, and 3 (6.1%) to genotype 4. Total 77.6% of TTV isolates of blood donors were deduced to be TTV type 1. 3.6. Serum ALT levels and TTV genotypes Elevated ALT level was found in 11 sera with 6 (27.3%) of type 1a, 2 (15.4%) of type 1b, and 3 (25%) of non-type 1 cases. No signi®cant dierence was observed in the rates for ALT level abnormality between genotype 1 and non-genotype 1 individuals. 3.7. Factors associated with TTV viremia Based on the results of multiple logistic regression analysis of positive TTV DNA, two factors including increased age and abnormal ALT levels were identi®ed to be independently associated with
C.-Y. Dai et al. / Transfusion and Apheresis Science 24 (2001) 9±15
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Table 2 Stepwise logistic regression analysis of factors signi®cantly associated with TTV DNA positivity in blood donors Dependent variable TTV DNA Positive
a
Independent variable
Comparison
Odds ratio (95% C.I.a )
Age ALT
Each year-old increase Abnormal vs. normal
1.03 (1.01±1.06) 1.9 (1.02±3.52)
C.I.: con®dence interval.
positive TTV DNA, as odds ratios and their corresponded 95% con®dence intervals are shown in Table 2. 4. Discussion The global distribution of TTV has been documented with a great variety of prevalence rates in dierent countries. TTV viremia can be detected by PCR methods using primers deduced from N22 region [9]. The TTV prevalences in blood donors by dierent primers were 1.9% in UK [11], 12± 81.7% in Japan [9,12,24], 62% in Brazil [20], 28% in China [28], and 1% in North America [29]. We report here that the prevalence of TTV DNA by N22 region primers in the southern Taiwanese blood donors negative for HBsAg and Anti-HCV is 23.4%. This result is higher than 11% from eastern Taiwan aboringines [19] and between 10% and 53% from northern Taiwan healthy subjects [22,23]. The clinical implication of TTV infection and etiological importance of TTV in association with liver diseases still remain unclear. After excluding the in¯uence of HBV or HCV infection, our study indicated that elevated ALT levels were associated with TTV infection. With the initial ®nding of TTV infection in posttransfusion hepatitis [8], TTV infection was suggested to cause hepatitis with elevated ALT levels [8,16,17]. Nevertheless, other reports revealed TTV infected subjects had normal ALT levels [19±21]. These dierent results may be explained by the possibility of a healthy carrier state of TTV infection [21]. Besides, when we consider the association between TTV infection and elevated ALT levels, two possible explanations are made: (1) TTV may really cause abnormality of liver function test, (2) people with elevated ALT
levels may be under the special circumstances and have higher rates of TTV infection. The prevalence of GBV-C/HGV RNA in our volunteer donors was 2.9% which was similar to the other reports of 0.9±3% in dierent parts of the world [2,30±32] and reports of 2.1% from Taiwan [33]. In our study, TTV viremia failed to show association with HGV exposure or viremia. We attributed the results to the lack of recovery markers for TTV infection to indicate the real exposure of TTV as anti-E2 in GBV-C/HGV infection [34,35]. The transmission of GBV-C/HGV was mainly through high-risk procedures or lifestyles, such as blood transfusion [2,36], intravenous drug use or sexual contact [37]. Our results may also indicate other important transmission routes dierent from GBV-C/HGV, such as the fecal-oral route in TTV infection [38,39]. There is a gradual increase in TTV infection rates with age in TTV infection groups shown in our study. With the similar previous reports for newborn to aged population [22,39], our results support that TTV has non-parenteral transmission routes and there is persistent TTV viremia [21]. Repeated donations did not decrease the TTV viremia rate in blood donors. This depicts the fact that available screening tests nowadays for donated blood cannot prevent TTV infection entirely. In the present study, the genetic heterogeneity of isolates cloned from southern Taiwanese donors existed by phylogenetic analysis of nucleotide sequence from the TTV genome. TTV genotype 1, which is distributed widely and most prevalent over the world, is also the most prevalent type among TTV isolates in our study as in previous reports from northern Taiwan [23] and Japan [24]. Genotype 1 had been reported to be associated with more pathogenetic potential [29]. We did not ®nd a signi®cant relationship between abnormal
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